Fixative and staining solutions

ABSTRACT

The formulations, systems, and methods disclosed herein permit automated preparation of specimens (e.g., biological specimens) for examination. The disclosed formulations, systems, and methods provide fast, efficient, and highly uniform specimen processing using minimal quantities of fluids. The methods include at least a fixing phase for fixing a specimen to a substrate such as a microscope slide, a staining phase for staining the specimen, and a rinsing phase for rinsing the specimen. One or more of the fixing, staining, and rinsing phases include one or more agitation phases for distributing reagents evenly and uniformly across the specimen. The systems can be implemented as a standalone device or as a component in a larger system for preparing and examining specimens.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.13/526,164, filed on Jun. 18, 2012, which claims the benefit of U.S.Provisional Application No. 61/498,159, filed on Jun. 17, 2011, U.S.Provisional Application No. 61/505,011, filed on Jul. 6, 2011, and U.S.Provisional Application No. 61/510,180, filed on Jul. 21, 2011; thedisclosures of each of which are incorporated herein by reference intheir entireties.

TECHNICAL FIELD

This disclosure relates to formulations for preparation of biologicalspecimens, and more particularly to fixing, staining, and rinsingformulations.

BACKGROUND

For years, laboratory technologists have used dyes and stains such asthose used in Romanowsky staining for preparing biological specimens toimprove the contrast of a specimen during examination. Such examinationtypically utilizes a microscope, an automated device that capturesimages of the specimen, or, in other instances, unaided visualexamination. Several different systems and methods for preparing aspecimen for examination are known. For example, U.S. Pat. Nos.6,096,271; 7,318,913; 5,419,279; and 5,948,360, and published U.S.Patent Application Nos. 2008/0102006 and 2006/0073074 relate to machinesand methods for staining a substrate during specimen processing. Thesepublications provide various details on staining and preparing specimensfor examination.

SUMMARY

The present disclosure relates to formulations, systems, and methods forpreparing specimens for examination. The specimens can include, forexample, blood samples comprising red blood cells, white blood cells,and platelets from biological samples, e.g., a blood sample, applied toa substrate, e.g., a microscope slide or a cover slip. Differentembodiments can be used to prepare specimens from biological samplesincluding bone marrow, urine, vaginal tissue, epithelial tissue, tumors,semen, saliva, and other body fluids. Additional aspects of thedisclosure include systems and methods for fixing, staining, rinsing,and agitating the specimens, using the formulations of the disclosure.In general, the formulations, systems, and methods disclosed hereinprovide for rapid, efficient, and highly uniform specimen preparationand processing using minimal fluid quantities. The methods include oneor more fixing, staining, and rinsing phases, including one or multipleagitation phases after one or more, e.g., after each of, the fixing,staining, and rinsing phases. The systems can be implemented as astandalone device or as a component in a larger system for preparing andexamining specimens.

In a first aspect, the disclosure features a cytological fixativesolution including Azure B; a surfactant; methanol; and ethylene glycol.

For example, the cytological fixative solution can include about 0.5 g/Lto about 5.0 g/L Azure B; about 0.5 mL/L to about 2.0 mL/L polysorbate20; about 5 mL/L to about 50 mL/L ethylene glycol, propylene glycol, orpolypropylene glycol; about 0.1 g/L to about 10 g/L HEPES sodium salt;and methanol.

As another example, the cytological fixative solution can include about0.8 g/L to about 1.2 g/L Azure B; about 0.8 mL/L to about 1.2 mL/Lpolysorbate 20; about 9 mL/L to about 11 mL/L ethylene glycol; about0.25 g/L to about 0.38 g/L HEPES sodium salt; and methanol.

For example, the cytological fixative solution can include about one g/LAzure B; about one mL/L polysorbate 20; about 10 mL/L ethylene glycol;about 0.32 g/L HEPES sodium salt; and methanol.

As another example, the cytological fixative solution can include aboutone g/L Azure B; about 0.5 mL/L polysorbate 20; about 10 mL/L propyleneglycol; about 0.32 g/L HEPES sodium salt; and methanol.

As a further example, the cytological fixative solution can includeabout one g/L Azure B; about 1.5 mL/L polysorbate 20; about 10 mL/Lpolypropylene glycol; about 0.32 g/L HEPES sodium salt; and methanol.

In a second aspect, the disclosure features a first cytological stainingsolution, including eosin Y; a buffering agent; a surfactant; sodiumchloride; ethylene glycol; and water.

For example, the first cytological staining solution can include about0.5 g/L to about 5.0 g/L Eosin Y; about 5 mM to about 250 mM bis-tris orphosphate buffer; about 0.5 mL/L to about 2.0 mL/L polysorbate 20; aboutone g/L to about 20 g/L sodium chloride; about five mL/L to about 50mL/L ethylene glycol; about 0.2 ppm to about 50 ppm ProClin 300®; aceticacid; and water, wherein the solution has a pH of from about 5.8 toabout 6.2.

As another example, the first cytological staining solution can includeabout 0.6 g/L to about 0.9 g/L Eosin Y; about 45 mM to about 55 mMbis-tris buffer; about 0.8 mL/L to about 1.2 mL/L polysorbate 20; aboutthree g/L to about five g/L sodium chloride; about nine mL/L to about 11mL/L ethylene glycol; about 10 ppm to about 20 ppm ProClin 300®; aceticacid; and water, wherein the solution has a pH of from about 5.8 toabout 6.2.

For example, the first cytological staining solution can include about0.75 g/L Eosin Y; about 50 mM bis-tris buffer; about one mL/Lpolysorbate 20; about four g/L sodium chloride; about 10 mL/L ethyleneglycol; about 15 ppm ProClin 300®; acetic acid; and water, wherein thesolution has a pH of from about 5.8 to about 6.2.

As another example, the first cytological staining solution can includeabout 0.75 g/L Eosin Y; about 50 mM bis-tris buffer; about 0.5 mL/Lpolysorbate 20; about six g/L sodium chloride; about 20 mL/L ethyleneglycol; about 15 ppm ProClin 300®; acetic acid; and water, wherein thesolution has a pH of from about 5.8 to about 6.2.

As a further example, the first cytological staining solution caninclude about 0.75 g/L Eosin Y; about 50 mM phosphate buffer; about 2.0mL/L polysorbate 20; about four g/L sodium chloride; about 50 mL/Lethylene glycol; about 15 ppm ProClin 300®; acetic acid; and water,wherein the solution has a pH of from about 5.8 to about 6.2.

In a third aspect, the disclosure features a second cytological stainingsolution, including Azure B; methylene blue; a buffering agent; asurfactant, sodium chloride, and water.

For example, the second cytological staining solution can include about0.25 to about 2.5 g/L Azure B; about 0.25 g/L to about 2.5 g/L methyleneblue; about 5 mM to about 250 mM bis-tris or HEPES buffer; about 0.5mL/L to about 2.0 mL/L polysorbate 20, about one g/L to about 20 g/Lsodium chloride, and about 0.2 ppm to about 50 ppm ProClin 300®; aceticacid; and water, wherein the solution has a pH of from about 6.8 toabout 7.2.

As another example, the second cytological staining solution can includeabout 0.4 to about 0.6 g/L Azure B; about 0.4 g/L to about 0.5 g/Lmethylene blue; about 45 mM to about 55 mM bis-tris buffer; about 0.8mL/L to about 1.2 mL/L polysorbate 20, about 1.8 g/L to about 2.2 g/Lsodium chloride, and about 10 ppm to about 20 ppm ProClin 300®; aceticacid; and water, wherein the solution has a pH of from about 6.8 toabout 7.2.

For example, the second cytological staining solution can include about0.5 g/L Azure B; about 0.45 g/L methylene blue; about 50 mM bis-trisbuffer; about one mL/L polysorbate 20, about two g/L sodium chloride,and about 15 ppm ProClin 300®; acetic acid; and water, wherein thesolution has a pH of from about 6.8 to about 7.2.

As another example, the second cytological staining solution can includeabout 0.5 g/L Azure B; about 0.45 g/L methylene blue; about 50 mM HEPESbuffer; about 0.5 mL/L polysorbate 20, about two g/L sodium chloride,and about 15 ppm ProClin 300®; acetic acid; and water, wherein thesolution has a pH of from about 6.8 to about 7.2.

As a further example, the second cytological staining solution caninclude about 0.5 g/L Azure B; about 0.45 g/L methylene blue; about 50mM HEPES buffer; about one mL/L polysorbate 20, about one g/L sodiumchloride, and about 15 ppm ProClin 300®; acetic acid; and water, whereinthe solution has a pH of from about 6.8 to about 7.2.

In a fourth aspect, the disclosure features a rinse solution for anautomated specimen preparation apparatus, including polyethylene glycol;a buffering agent; a surfactant; methanol; and water.

For example, the rinse solution can include about 0.2 g/L to about teng/L polyethylene glycol; about 1 mM to about 250 mM HEPES, MES, orbis-tris buffer; about 0.1 mL/L to about 2.40 mL/L polysorbate 20; about0.04 ppm to about 50 ppm ProClin 300®; about 9 mL/L to about 200 mL/Lmethanol; and water, wherein the rinse solution has a pH of from about6.6 to about 7.0.

For example, the rinse solution can include about one g/L to about teng/L polyethylene glycol; about 5 mM to about 250 mM HEPES, MES, orbis-tris buffer; about 0.5 mL/L to about 2.0 mL/L polysorbate 20; about0.2 ppm to about 50 ppm ProClin 300®; about 10 mL/L to about 200 mL/Lmethanol; and water, wherein the rinse solution has a pH of from about6.6 to about 7.0.

As another example, the rinse solution can include about 4.5 g/L toabout 5.5 g/L polyethylene glycol; about 45 mM to about 55 mM HEPESbuffer; about 0.8 mL/L to about 1.2 mL/L polysorbate 20; about 10 ppm toabout 20 ppm ProClin 300®; about 45 mL/L to about 55 mL/L methanol; andwater, wherein the rinse solution has a pH of from about 6.6 to about7.0.

For example, the rinse solution includes about five g/L polyethyleneglycol; about 50 mM HEPES buffer; about one mL/L polysorbate 20; about15 ppm ProClin 300®; about 50 mL/L methanol; and water, wherein therinse solution has a pH of from about 6.6 to about 7.0.

As another example, the rinse solution includes about 10 g/Lpolyethylene glycol; about 50 mM MES buffer; about one mL/L polysorbate20; about 15 ppm ProClin 300®; about 50 mL/L methanol; and water,wherein the rinse solution has a pH of from about 6.6 to about 7.0.

As a further example, the rinse solution includes about 10 g/Lpolyethylene glycol; about 50 mM bis-tris buffer; about 0.5 mL/Lpolysorbate 20; about 15 ppm ProClin 300®; about 50 mL/L methanol; andwater, wherein the rinse solution has a pH of from about 6.6 to about7.0.

For example, the rinse solution can include about 0.2 g/L to about twog/L polyethylene glycol; about one mM to about 50 mM HEPES buffer; about0.16 mL/L to about 0.24 mL/L polysorbate 20; about 0.04 ppm to about 10ppm ProClin 300®; about 9 mL/L to about 11 mL/L methanol; and water,wherein the rinse solution has a pH of from about 6.6 to about 7.0.

As another example, the rinse solution can include about 0.9 g/L toabout 1.1 g/L polyethylene glycol; about 9 mM to about 11 mM HEPESbuffer; about 0.16 mL/L to about 0.24 mL/L polysorbate 20; about 2 ppmto about 10 ppm ProClin 300®; about 9 mL/L to about 11 mL/L methanol;and water, wherein the rinse solution has a pH of from about 6.6 toabout 7.0.

In still another example, the rinse solution includes about one g/Lpolyethylene glycol; about 10 mM HEPES buffer; about 0.2 mL/Lpolysorbate 20; about 3 ppm ProClin 300®; about 10 mL/L methanol; andwater, wherein the rinse solution has a pH of from about 6.6 to about7.0.

In a fifth aspect, the disclosure features a specimen preparation kitincluding a cytological fixative solution; a first cytological stainingsolution; a second cytological staining solution; and a rinse solution.In some embodiments, the specimen preparation kit includes a separatelypackaged cytological fixative solution; a separately packaged firstcytological staining solution; a separately packaged second cytologicalstaining solution; and a separately packaged rinse solution.

In a sixth aspect, the disclosure features a method of preparing aspecimen on a substrate for examination, including (a) positioning thesubstrate with respect to a surface so that the specimen faces thesurface, so that the substrate is positioned to form a separationbetween the surface and at least a portion of the substrate of at leastabout 100 microns; (b) performing a fixing phase including (i)dispensing a fixative solution into the separation between the substrateand the surface in an amount sufficient to contact the specimen and thesurface; (ii) performing at least a first agitation phase, wherein thefirst agitation phase comprises changing the distance between thesubstrate and surface while the fixative solution is contacting thespecimen for the duration of the first agitation phase; and (iii)removing the fixative solution from the separation and the specimen; (c)performing a first staining phase; (d) performing a second stainingphase; and (e) performing a first rinse phase.

In a seventh aspect, the disclosure features a system for imaging aspecimen, including an automated specimen analysis machine, and acytological fixative solution; a first cytological staining solution; asecond cytological staining solution; and/or a rinse solution. In someembodiments, the system for imaging a specimen includes an automatedspecimen analysis machine and a specimen prepared with: a cytologicalfixative solution; a first cytological staining solution; a secondcytological staining solution; and/or a rinse solution.

Embodiments of the cytological fixative solutions, first cytologicalstaining solutions, second cytological staining solutions, rinsesolutions, methods, kits, and systems described herein can include anyone or more of the following features.

In some embodiments, the cytological fixative solution can include fromabout 0.5 to about five g/L of Azure B (e.g., about one g/L Azure B).For example, a 1:1000 dilution of the cytological fixative solution inwater has a UV absorbance of from about 0.1 to about one at a peakwavelength of from about 640 to about 650 nm.

In some embodiments, the cytological fixative solution is free orsubstantially free of one (or more) red stains (e.g., one or moreconventional red stains known in the art, e.g., eosin Y, and fluoresceinderivatives).

The cytological fixative solution can include from about 0.05% to about0.5% by volume or by weight (e.g., from about 0.05% to about 0.3% byvolume or by weight of the surfactant, from about 0.05% to about 0.1% byvolume or by weight) of the surfactant. The surfactant can be selectedfrom the group consisting of non-ionic, cationic, anionic, andzwitterionic surfactants. In some embodiments, the surfactant isnon-ionic, and can include, for example, polysorbate 20. For example,the cytological fixative solution can include from about 0.5 mL/L toabout two mL/L (e.g., from about 0.5 mL/L to about 1.5 mL/L, about oneml/L) of polysorbate 20.

In some embodiments, the cytological fixative solution can furtherinclude a buffering agent, such as bis-tris, phosphate, HEPES, MES,and/or Tris buffers. For example, a 1:10 dilution of the cytologicalfixative solution in water can have a pH of from about 6 to about 8(e.g., from about 6.7 to about 7.3) and include from about 0.5 mM toabout 10 mM of HEPES.

The cytological fixative solution can include from about 0.5 to about 5%(e.g., about one percent) by volume ethylene glycol.

In some embodiments, the cytological fixative solution can include aboutone g/L Azure B; about one mL/L polysorbate 20; about 10 mL/L ethyleneglycol; about 0.32 g/L HEPES sodium salt; and methanol.

In some embodiments, the first cytological staining solution can includean antimicrobial agent (e.g., from about 0.2 to about 50 ppm of theantimicrobial agent), which can include, for example, benzalkoniumchloride, 5-chloro-2-methyl-4-isothiazolin-3-one,2-methyl-4-isothiazolin-3-one, ProClin® (e.g., ProClin 300®), azides,merthiolates, antibiotics, and any combination thereof. For example, theantimicrobial agent can include 5-chloro-2-methyl-4-isothiazolin-3-oneand 2-methyl-4-isothiazolin-3-one. In some embodiments, theantimicrobial agent is ProClin 300®. For example, the first cytologicalstaining solution can include about 15 ppm ProClin 300®.

The first cytological staining solution can include from about 0.5 toabout one g/L (e.g., about 0.75 g/L) eosin Y. For example, a 1:500dilution of the solution in water can have a UV absorbance of from about0.1 to about one at a peak wavelength of from about 510 to about 530 nm.

In some embodiments, the first cytological staining solution is free orsubstantially free of one (or more) other red stains (e.g., one or moreother conventional red stains known in the art, e.g., other fluoresceinderivatives).

In some embodiments, the first cytological staining solution is free orsubstantially free of one (or more) blue stains (e.g., one or moreconventional blue stains known in the art, e.g., azure B, methyleneblue, and/or other thiazine dyes).

In some embodiments, the first cytological staining solution is free orsubstantially free of one (or more) other red stains (e.g., one or moreother conventional red stains known in the art, e.g., other fluoresceinderivatives), and the first cytological staining solution is free orsubstantially free of one (or more) blue stains (e.g., one or moreconventional blue stains known in the art, e.g., azure B, methyleneblue, and/or other thiazine dyes).

The first cytological staining solution can include from about 0.5 toabout five percent (e.g., about one percent) by volume ethylene glycol.

In some embodiments, the first cytological staining solution can have apH of from about 5 to about 8 and a buffering agent concentration offrom about five to about 250 mM.

The buffering agent can include bis-tris, phosphate, HEPES, MES, Tris,and any combination thereof. For example, the first cytological stainingsolution can have a pH of from about 5.8 to about 6.2 and a bis-trisbuffer concentration of from about five mM to about 250 mM. In someembodiments, the first cytological staining solution can further includeacetic acid (e.g., from about two to about three mL/L acetic acid).

In some embodiments, the first cytological staining solution can includefrom about 0.05 to about 0.5% (e.g., from about 0.05% to about 0.3%,from about 0.05% to about 0.1%) by volume or by weight of thesurfactant. The surfactant can be selected from the group consisting ofnon-ionic, cationic, anionic or zwitterionic surfactants. For example,the surfactant can be non-ionic, such as polysorbate 20. In someembodiments, the first cytological staining solution can include fromabout 0.5 mL/L to about two mL/L (e.g., from about 0.5 mL/L to about 1.5mL/L, about one ml/L) of polysorbate 20.

In some embodiments, the first cytological staining solution can includefrom about one to about 20 g/L sodium chloride.

In some embodiments, the first cytological staining solution can includeabout 0.75 g/L Eosin Y; about 50 mM bis-tris buffer; about one mL/Lpolysorbate 20; about four g/L sodium chloride; about 10 mL/L ethyleneglycol; about 15 ppm ProClin 300®; acetic acid; and water, wherein thesolution has a pH of from about 5.8 to about 6.2.

In some embodiments, the second cytological staining solution caninclude an antimicrobial agent (e.g., from about 0.2 to about 50 ppm ofthe antimicrobial agent), which can include, for example, benzalkoniumchloride, 5-chloro-2-methyl-4-isothiazolin-3-one,2-methyl-4-isothiazolin-3-one, ProClin® (e.g., ProClin 300®), azides,merthiolates, antibiotics, and any combination thereof. For example, theantimicrobial agent can include 5-chloro-2-methyl-4-isothiazolin-3-oneand 2-methyl-4-isothiazolin-3-one. In some embodiments, the secondcytological staining solution can include ProClin 300® (e.g., about 15ppm ProClin 300®).

In some embodiments, the second cytological staining solution caninclude from about 0.25 to about one g/L (e.g., about 0.5 g/L) azure B.The second cytological staining solution can include from about 0.25 toabout one g/L (e.g., about 0.45 g/L) methylene blue. For example, a1:1000 dilution of the second cytological staining solution in water canhave a UV absorbance of from about 0.1 to about one at a peak wavelengthof from about 640 to about 660 nm.

In some embodiments, the second cytological staining solution is free orsubstantially free of one (or more) other blue stains (e.g., one or moreother conventional blue stains known in the art, e.g., azure B,methylene blue, and/or other thiazine dyes).

In some embodiments, the second cytological staining solution is free orsubstantially free of one (or more) red stains (e.g., one or moreconventional red stains known in the art, e.g., fluoresceinderivatives).

In some embodiments, the second cytological staining solution is free orsubstantially free of one (or more) other blue stains (e.g., one or moreother conventional blue stains known in the art, e.g., other thiazinedyes), and the second cytological staining solution is free orsubstantially free of one (or more) red stains (e.g., one or moreconventional red stains known in the art, e.g., fluoresceinderivatives).

In some embodiments, the second staining solution can have a pH of fromabout 5 to about 8 and a buffering agent concentration of from about 5mM to about 250 mM (e.g., a pH of from about 6.8 to about 7.2 and abis-tris buffer concentration of from about 25 mM to about 100 mM). Thebuffering agent can include bis-tris buffer, phosphate, HEPES, MES,Tris, and any combination thereof. In some embodiments, the secondcytological staining solution can further include acetic acid.

In some embodiments, the second cytological staining solution caninclude from about 0.05 to about 0.5% (e.g., from about 0.05% to about0.3%, from about 0.05% to about 0.1%) by volume or by weight of thesurfactant, which can include non-ionic, cationic, anionic, andzwitterionic surfactants. In some embodiments, the surfactant isnon-ionic and can include polysorbate 20, and the second cytologicalstaining solution can include from about 0.5 mL/L to about two mL/L(e.g., from about 0.5 mL/L to about 1.5 mL/L, about one ml/L) ofpolysorbate 20.

In some embodiments, the second cytological staining solution caninclude from about one to about 20 g/L sodium chloride.

In some embodiments, the second cytological staining solution caninclude about 0.5 g/L Azure B; about 0.45 g/L methylene blue; about 50mM bis-tris buffer; about one mL/L polysorbate 20, about two g/L sodiumchloride, and about 15 ppm ProClin 300®; acetic acid; and water. Thesolution can have a pH of from about 6.8 to about 7.2.

In some embodiments, the rinse solution can include an antimicrobialagent (e.g., from about 0.2 to about 50 ppm of the antimicrobial agentor from about 0.04 to about 10 ppm of the antimicrobial agent), whichcan include, for example, benzalkonium chloride,5-chloro-2-methyl-4-isothiazolin-3-one, 2-methyl-4-isothiazolin-3-one,ProClin® (e.g., ProClin 300®), azides, merthiolates, antibiotics, andany combination thereof. In some embodiments, the antimicrobial agentcan include 5-chloro-2-methyl-4-isothiazolin-3-one and2-methyl-4-isothiazolin-3-one. For example, the rinse solution caninclude ProClin 300® (e.g., about 15 ppm ProClin 300 to about 3 ppmProClin 300°).

The rinse solution can include from about one to about ten g/L (e.g.,about five g/L) polyethylene glycol.

The rinse solution can include from about 0.2 to about two g/L (e.g.,about one g/L) polyethylene glycol.

The rinse solution can have a pH of from 5 to 8 and a buffering agentconcentration of from about five mM to about 250 mM (e.g., a pH of fromabout 5 to about 8 and a HEPES buffer concentration of about 50 mM). Thebuffering agent can include bis-tris buffer, phosphate, HEPES, MES,Tris, and any combination thereof.

The rinse solution can have a pH of from 5 to 8 and a buffering agentconcentration of from about one mM to about 50 mM (e.g., a pH of fromabout 5 to about 8 and a HEPES buffer concentration of about 10 mM). Thebuffering agent can include bis-tris buffer, phosphate, HEPES, MES,Tris, and any combination thereof.

In some embodiments, the rinse solution includes from about 0.05 toabout 0.5% (e.g., from about 0.05% to about 0.3%, from about 0.05% toabout 0.1%) by volume or by weight of the surfactant. The surfactant canbe selected from the group consisting of non-ionic, cationic, anionic,and zwitterionic surfactants. For example, the surfactant can benon-ionic and can include polysorbate 20 (e.g., from about 0.5 mL/L toabout two mL/L, from about 0.5 mL/L to about 1.5 mL/L of polysorbate20).

In some embodiments, the rinse solution includes from about 0.01 toabout 0.1% (e.g., from about 0.01% to about 0.06%, from about 0.01% toabout 0.02%) by volume or by weight of the surfactant. The surfactantcan be selected from the group consisting of non-ionic, cationic,anionic, and zwitterionic surfactants. For example, the surfactant canbe non-ionic and can include polysorbate 20 (e.g., from about 0.1 mL/Lto about 0.4 mL/L, from about 0.1 mL/L to about 0.3 mL/L of polysorbate20).

In some embodiments, the rinse solution includes from about 45 to about55 mL/L (e.g., about 50 mL/L) methanol.

In some embodiments, the rinse solution includes from about 9 to about11 mL/L (e.g., about 10 mL/L) methanol.

In some embodiments, the rinse solution includes about five g/Lpolyethylene glycol; about 50 mM HEPES buffer; about one mL/Lpolysorbate 20; about 15 ppm ProClin 300®; about 50 mL/L methanol; andwater. The rinse solution can have a pH of from about 6.6 to about 7.0.

In some embodiments, the rinse solution includes about one g/Lpolyethylene glycol; about 10 mM HEPES buffer; about 0.2 mL/Lpolysorbate 20; about 3 ppm ProClin 300®; about 10 mL/L methanol; andwater. The rinse solution can have a pH of from about 6.6 to about 7.0.

In some embodiments, the cytological fixative solutions are free of thedye Patent Blue VF (e.g., contain none or less than the amount of PatentBlue VF that is present in the formulations disclosed in WO2007/117798).

In some embodiments, first cytological staining solutions are free ofthe dye Patent Blue VF (e.g. contain none or less than the amount ofPatent Blue VF that is present in the formulations disclosed in WO2007/117798).

In some embodiments, second cytological staining solutions are free ofthe dye Patent Blue VF (e.g. contain none or less than the amount ofPatent Blue VF that is present in the formulations disclosed in WO2007/117798).

In some embodiments, the rinse solutions are free of the dye Patent BlueVF (e.g. contain none or less than the amount of Patent Blue VF that ispresent in the formulations disclosed in WO 2007/117798).

In some embodiments, the cytological fixative solutions are free of aproteolytic enzyme (e.g., contain none or less than the amount of aproteolytic enzyme that is present in the formulations disclosed in WO2007/117798).

In some embodiments, first cytological staining solutions are free of aproteolytic enzyme (e.g., contain none or less than the amount of aproteolytic enzyme that is present in the formulations disclosed in WO2007/117798).

In some embodiments, second cytological staining solutions are free of aproteolytic enzyme (e.g., contain none or less than the amount of aproteolytic enzyme that is present in the formulations disclosed in WO2007/117798).

In some embodiments, the rinse solutions are free of a proteolyticenzyme (e.g., contain none or less than the amount of a proteolyticenzyme that is present in the formulations disclosed in WO 2007/117798).

In some embodiments, performing a first staining phase includes (i)dispensing a first cytological staining solution into the separationbetween the substrate and the surface in an amount sufficient to contactthe specimen and the surface; (ii) performing at least a secondagitation phase, wherein the second agitation phase includes changingthe distance between the substrate and surface while the first stainingsolution is contacting the specimen for the duration of the secondagitation phase; and (iii) removing the first staining solution from theseparation and the specimen.

In some embodiments, performing a second staining phase includes (i)dispensing a second cytological staining solution into the separationbetween the substrate and the surface in an amount sufficient to contactthe specimen and the surface; (ii) performing at least a third agitationphase, wherein the third agitation phase comprises changing the distancebetween the substrate and surface while the second staining solution iscontacting the specimen for the duration of the third agitation phase;and (iii) removing the second staining solution from the separation andthe specimen.

In some embodiments, performing a first rinse phase includes (i)dispensing a rinse solution into the separation between the substrateand the surface in an amount sufficient to contact the specimen and thesurface; (ii) performing at least a fourth agitation phase, wherein thefourth agitation phase comprises changing the distance between thesubstrate and surface while the rinse solution is contacting thespecimen for the duration of the fourth agitation phase; and (iii)removing the rinse solution from the separation and the specimen.

In some embodiments, the automated specimen analysis machine includes asubstrate arm including a substrate gripper; a first actuator connectedto the substrate arm and configured to move the substrate arm between anopen position and a specimen processing position; a second actuatorarranged and configured to agitate a substrate gripped by the substrategripper on the substrate arm; a platform having a top surface locatedopposite the substrate when the substrate arm is in the specimenprocessing position; and two or more offsets arranged on the top surfaceof the platform such that when the substrate contacts all of the offsetsin the substrate processing position, the substrate and top surface ofthe platform are substantially parallel and form a separation of atleast about 50 microns.

Embodiments of the automated specimen examination system can include anyone or more of the features disclosed herein, as appropriate, includingany one or more of the features of the specimen preparation apparatus'disclosed herein.

In embodiments of the methods, kits, and systems described herein, one,two, three, or four (e.g., four) of (A), (B), (C), and (D) can apply.

(A) The cytological fixative solution includes Azure B; a surfactant;methanol; and ethylene glycol.

For example, the cytological fixative solution can include about 0.5 g/Lto about 5.0 g/L Azure B; about 0.5 mL/L to about 2.0 mL/L polysorbate20; about 5 mL/L to about 50 mL/L ethylene glycol, propylene glycol, orpolypropylene glycol; about 0.1 g/L to about 10 g/L HEPES sodium salt;and methanol.

As another example, the cytological fixative solution can include about0.8 g/L to about 1.2 g/L Azure B; about 0.8 mL/L to about 1.2 mL/Lpolysorbate 20; about 9 mL/L to about 11 mL/L ethylene glycol; about0.25 g/L to about 0.38 g/L HEPES sodium salt; and methanol.

For example, the cytological fixative solution can include about one g/LAzure B; about one mL/L polysorbate 20; about 10 mL/L ethylene glycol;about 0.32 g/L HEPES sodium salt; and methanol.

As another example, the cytological fixative solution can include aboutone g/L Azure B; about 0.5 mL/L polysorbate 20; about 10 mL/L propyleneglycol; about 0.32 g/L HEPES sodium salt; and methanol.

As a further example, the cytological fixative solution can includeabout one g/L Azure B; about 1.5 mL/L polysorbate 20; about 10 mL/Lpolypropylene glycol; about 0.32 g/L HEPES sodium salt; and methanol.

Embodiments can include any one or more of the features described aboveand/or in the Detailed Description and/or in the claims.

(B) The first cytological staining solution includes eosin Y; abuffering agent; a surfactant; sodium chloride; ethylene glycol; andwater.

For example, the first cytological staining solution can include about0.5 g/L to about 5.0 g/L Eosin Y; about 5 mM to about 250 mM bis-tris orphosphate buffer; about 0.5 mL/L to about 2.0 mL/L polysorbate 20; aboutone g/L to about 20 g/L sodium chloride; about five mL/L to about 50mL/L ethylene glycol; about 0.2 ppm to about 50 ppm ProClin 300®; aceticacid; and water, wherein the solution has a pH of from about 5.8 toabout 6.2.

As another example, the first cytological staining solution can includeabout 0.6 g/L to about 0.9 g/L Eosin Y; about 45 mM to about 55 mMbis-tris buffer; about 0.8 mL/L to about 1.2 mL/L polysorbate 20; aboutthree g/L to about five g/L sodium chloride; about nine mL/L to about 11mL/L ethylene glycol; about 10 ppm to about 20 ppm ProClin 300®; aceticacid; and water, wherein the solution has a pH of from about 5.8 toabout 6.2.

For example, the first cytological staining solution can include about0.75 g/L Eosin Y; about 50 mM bis-tris buffer; about one mL/Lpolysorbate 20; about four g/L sodium chloride; about 10 mL/L ethyleneglycol; about 15 ppm ProClin 300®; acetic acid; and water, wherein thesolution has a pH of from about 5.8 to about 6.2.

As another example, the first cytological staining solution can includeabout 0.75 g/L Eosin Y; about 50 mM bis-tris buffer; about 0.5 mL/Lpolysorbate 20; about six g/L sodium chloride; about 20 mL/L ethyleneglycol; about 15 ppm ProClin 300®; acetic acid; and water, wherein thesolution has a pH of from about 5.8 to about 6.2.

As a further example, the first cytological staining solution caninclude about 0.75 g/L Eosin Y; about 50 mM phosphate buffer; about 2.0mL/L polysorbate 20; about four g/L sodium chloride; about 50 mL/Lethylene glycol; about 15 ppm ProClin 300®; acetic acid; and water,wherein the solution has a pH of from about 5.8 to about 6.2.

Embodiments can include any one or more of the features described aboveand/or in the Detailed Description and/or in the claims.

(C) The second cytological staining solution includes Azure B; methyleneblue; a buffering agent; a surfactant, sodium chloride, and water.

For example, the second cytological staining solution can include about0.25 to about 2.5 g/L Azure B; about 0.25 g/L to about 2.5 g/L methyleneblue; about 5 mM to about 250 mM bis-tris or HEPES buffer; about 0.5mL/L to about 2.0 mL/L polysorbate 20, about one g/L to about 20 g/Lsodium chloride, and about 0.2 ppm to about 50 ppm ProClin 300®; aceticacid; and water, wherein the solution has a pH of from about 6.8 toabout 7.2.

As another example, the second cytological staining solution can includeabout 0.4 to about 0.6 g/L Azure B; about 0.4 g/L to about 0.5 g/Lmethylene blue; about 45 mM to about 55 mM bis-tris buffer; about 0.8mL/L to about 1.2 mL/L polysorbate 20, about 1.8 g/L to about 2.2 g/Lsodium chloride, and about 10 ppm to about 20 ppm ProClin 300®; aceticacid; and water, wherein the solution has a pH of from about 6.8 toabout 7.2.

For example, the second cytological staining solution can include about0.5 g/L Azure B; about 0.45 g/L methylene blue; about 50 mM bis-trisbuffer; about one mL/L polysorbate 20, about two g/L sodium chloride,and about 15 ppm ProClin 300®; acetic acid; and water, wherein thesolution has a pH of from about 6.8 to about 7.2.

As another example, the second cytological staining solution can includeabout 0.5 g/L Azure B; about 0.45 g/L methylene blue; about 50 mM HEPESbuffer; about 0.5 mL/L polysorbate 20, about two g/L sodium chloride,and about 15 ppm ProClin 300®; acetic acid; and water, wherein thesolution has a pH of from about 6.8 to about 7.2.

As a further example, the second cytological staining solution caninclude about 0.5 g/L Azure B; about 0.45 g/L methylene blue; about 50mM HEPES buffer; about one mL/L polysorbate 20, about one g/L sodiumchloride, and about 15 ppm ProClin 300®; acetic acid; and water, whereinthe solution has a pH of from about 6.8 to about 7.2.

The solution can have a pH of from about 6.8 to about 7.2.

Embodiments can include any one or more of the features described aboveand/or in the Detailed Description and/or in the claims.

(D) The rinse solution for an automated specimen preparation apparatusincludes polyethylene glycol; a buffering agent; a surfactant; methanol;and water.

For example, the rinse solution can include about 0.2 g/L to about teng/L polyethylene glycol; about 1 mM to about 250 mM HEPES, MES, orbis-tris buffer; about 0.1 mL/L to about 2.40 mL/L polysorbate 20; about0.04 ppm to about 50 ppm ProClin 300®; about 9 mL/L to about 200 mL/Lmethanol; and water, wherein the rinse solution has a pH of from about6.6 to about 7.0.

As another example, the rinse solution can include about one g/L toabout ten g/L polyethylene glycol; about 5 mM to about 250 mM HEPES,MES, or bis-tris buffer; about 0.5 mL/L to about 2.0 mL/L polysorbate20; about 0.2 ppm to about 50 ppm ProClin 300®; about 10 mL/L to about200 mL/L methanol; and water, wherein the rinse solution has a pH offrom about 6.6 to about 7.0.

As a further example, the rinse solution can include about 4.5 g/L toabout 5.5 g/L polyethylene glycol; about 45 mM to about 55 mM HEPESbuffer; about 0.8 mL/L to about 1.2 mL/L polysorbate 20; about 10 ppm toabout 20 ppm ProClin 300®; about 45 mL/L to about 55 mL/L methanol; andwater, wherein the rinse solution has a pH of from about 6.6 to about7.0.

For example, the rinse solution includes about five g/L polyethyleneglycol; about 50 mM HEPES buffer; about one mL/L polysorbate 20; about15 ppm ProClin 300®; about 50 mL/L methanol; and water, wherein therinse solution has a pH of from about 6.6 to about 7.0.

As another example, the rinse solution includes about 10 g/Lpolyethylene glycol; about 50 mM MES buffer; about one mL/L polysorbate20; about 15 ppm ProClin 300®; about 50 mL/L methanol; and, wherein therinse solution has a pH of from about 6.6 to about 7.0.

As a further example, the rinse solution includes about 10 g/Lpolyethylene glycol; about 50 mM bis-tris buffer; about 0.5 mL/Lpolysorbate 20; about 15 ppm ProClin 300®; about 50 mL/L methanol; andwater, wherein the rinse solution has a pH of from about 6.6 to about7.0.

For example, the rinse solution can include about 0.2 g/L to about twog/L polyethylene glycol; about one mM to about 50 mM HEPES buffer; about0.16 mL/L to about 0.24 mL/L polysorbate 20; about 0.04 ppm to about 10ppm ProClin 300®; about 9 mL/L to about 11 mL/L methanol; and water,wherein the rinse solution has a pH of from about 6.6 to about 7.0.

As another example, the rinse solution can include about 0.9 g/L toabout 1.1 g/L polyethylene glycol; about 9 mM to about 11 mM HEPESbuffer; about 0.16 mL/L to about 0.24 mL/L polysorbate 20; about 2 ppmto about 10 ppm ProClin 300®; about 9 mL/L to about 11 mL/L methanol;and water, wherein the rinse solution has a pH of from about 6.6 toabout 7.0.

In still another example, the rinse solution includes about one g/Lpolyethylene glycol; about 10 mM HEPES buffer; about 0.2 mL/Lpolysorbate 20; about 3 ppm ProClin 300®; about 10 mL/L methanol; andwater, wherein the rinse solution has a pH of from about 6.6 to about7.0.

As used herein, when a solution is “free” of one or more substances,this means that the solution contain less than 5% (e.g., less than 4%,less than 3%, less than 2%, less than 1%, 0% (w/w or w/v or v/v)) of theindicated one or more substances as determined by HPLC, UV spectroscopy,electrophoresis, and/or enzyme assay detection.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In case of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

Other features and advantages of the invention will be apparent from thefollowing detailed description, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1 is an HPLC chromatograph of an embodiment of a fixative solution.

FIG. 2 is a UV-Vis absorption spectrum of an embodiment of a fixativesolution.

FIG. 3 is an HPLC chromatograph of an embodiment of a first stainingsolution.

FIG. 4 is a UV-Vis absorption spectrum of an embodiment of a firststaining solution.

FIG. 5 is an HPLC chromatograph of an embodiment of a second stainingsolution.

FIG. 6 is a UV-Vis absorption spectrum of an embodiment of a secondstaining solution.

FIG. 7 is a perspective view of an embodiment of an apparatus forpreparing specimens for examination, with both sample grippers 20A and20B in an open position.

FIG. 8 is another perspective view of a portion of the apparatus of FIG.7 (with the substrate arms and sample grippers not shown).

FIG. 9A is a further perspective view of the apparatus of FIG. 7, withsample gripper 20A in an open position and sample gripper 20B in aclosed (specimen processing) position.

FIG. 9B is a perspective view of an indexing mechanism of the apparatusof FIG. 7.

FIG. 10 is a perspective view of the apparatus of FIG. 7 showingconnections between the apparatus and fluid reservoirs by means ofmultiple fluid conduits.

FIG. 11 is a perspective view of a specimen examination system thatincludes an automated substrate mover and an embodiment of a specimenpreparation apparatus as described herein.

FIG. 12A is an expanded perspective view of a portion of the apparatusof FIG. 7 showing specimen gripper 20B, platform 60B, and block 80B indetail.

FIG. 12B is a perspective view of a ball joint mechanism of theapparatus of FIG. 7.

FIG. 12C is a cross-sectional view of the ball joint mechanism of FIG.12B.

FIG. 13A is a flow chart showing a series of steps for moving substratearms from an open position to closed (specimen processing) position.

FIG. 13B is a schematic diagram of an embodiment of a specimenpreparation apparatus as described herein.

FIG. 14A is a flow chart showing an alternate series of steps for movingsubstrate arms from an open position to a specimen processing position.

FIG. 14B is a schematic diagram of an apparatus for preparing specimensfor examination that includes two actuators.

FIG. 15 is a flow chart showing a series of steps for applying fixativeto a specimen.

FIG. 16 is a flow chart showing a series of steps for applying stain toa specimen.

FIG. 17A is a flow chart showing a series of steps for removing excessfluid from a substrate.

FIG. 17B is a flow chart showing an alternate series of steps forremoving excess fluid from a substrate.

FIG. 18 is a flow chart showing a series of steps for rinsing aspecimen.

FIG. 19 is a flow chart showing a series of steps for agitating aspecimen.

FIG. 20 is a flow chart showing a series of steps for drying a specimen.

FIG. 21 is a perspective view of a specimen preparation apparatus asused in a larger specimen examination system.

FIG. 22 is a flow chart showing a series of steps for processing aspecimen mounted on a substrate.

FIG. 23 is a graph showing volume of fluid consumed as a function oftime in the flow chart of FIG. 22.

FIGS. 24A and 24B are perspective views of the apparatus of FIG. 7 thatshow placement of a substrate onto a substrate arm by an automatedsubstrate mover.

FIG. 25 is an embodiment of a kit containing a fixative solution, afirst staining solution, a second staining solution, and a rinsesolution.

Like reference symbols in the various drawings indicate like elements.

DETAILED DESCRIPTION

Disclosed herein are formulations, methods, and systems for automatedspecimen processing. The automated specimen processing methods andsystems described herein provide advantages over manual and otherautomated processing methods, including enhanced processing speed usingminimal reagent volumes with a concurrent highly uniform staining thatsignificantly reduces the variability associated with the application ofstains, fixatives, and other reagents when specimens are processed byhand or by other systems. The fixative, stain, and rinse solutions canprovide enhanced contrast to the specimen, and can be easily dispensedfrom the automated specimen processing systems. Further, the fixative,stain, and rinse solutions can provide improved cell identification andcell classification over conventional fixative, stain, and rinsesolutions.

Conventional fixative, staining, and rinsing solutions typically providepoor staining results, such as light-colored staining or non-specificstaining of biological samples (e.g., non-specific staining ofcytoplasms); or require large volumes of processing fluids. Theformulations disclosed herein permit high contrast and selectivestaining of biological samples with low volumes of processing fluids,for example, when used with the automated specimen processing systems.

Furthermore, conventional automated processing methods typically haverelatively high processing throughput while at the same time consuminglarge volumes of processing fluids, or have relatively low processingthroughput while consuming reduced volumes of fluids. For manyapplications, however, both high throughput operation and low fluidconsumption are desirable. By maintaining high throughput, specimens canbe efficiently processed and examined. By keeping fluid consumption low,the amount of processing waste is reduced along with the required volumeof processing reagents, keeping operating costs low. The formulations,systems, and methods disclosed herein permit rapid automated processingof specimens (e.g., more than 100 specimens per hour by a singlemachine) using low volumes of processing fluids (e.g., less than about 1mL of fluids per specimen, less than about 1.5 mL of fluids perspecimen, less than about 2 mL of fluids per specimen), while producinghighly uniform and repeatable results.

Formulations for Use with Specimen Preparation Systems

A fixative solution, one or more staining solutions, and a rinsesolution are provided herein, which can be used in an automated specimenpreparation system. Each of the solutions can have a specificformulation. For example, the fixative solution can include acytological dye, a surfactant, an organic solvent, an agent to minimizenon-specific binding, and a buffering agent. A first aqueous stainingsolution can include a cytological dye, a buffering agent, a surfactant,a salt, an agent to minimize non-specific binding, and an antimicrobialagent. A second aqueous staining solution can include one or morecytological dye(s), a buffering agent, a surfactant, a salt, and anantimicrobial agent. The rinse solution can include a buffering agent, asurfactant, an antimicrobial, an organic solvent, and a water-solublepolymer. The formulation components can be analyzed for purity and/orpurified by HPLC. The formulations can also include an acid foradjusting the pH. Each formulation is described in greater detail below.

Fixative Solution

Fixatives that can be included in a fixative solution include chemicalsused for protecting biological samples from decay. Such fixatives canimpede biochemical reactions occurring in the specimen and increase themechanical strength and stability of the specimen. Various fixatives canbe included in a fixative solution, e.g., methanol, ethanol,isopropanol, acetone, formaldehyde, glutaraldehyde, EDTA, surfactants,metal salts, metal ions, urea, and amino compounds. In some embodiments,the fixative solution includes an organic solvent or a fixation agentthat can precipitate, crosslink, or otherwise preserve components (e.g.,proteins) within a cell for imaging purposes. For example, the fixationagent can include an organic solvent, such as one or more alcoholsincluding methanol, ethanol, and isopropanol, or other organic solventsuch as acetone and ether. In some embodiments, the fixative solutionincludes an organic solvent such as methanol. The organic solvent canhave an ACS grade of less than about 0.1% water (e.g., less than about0.05% water, less than about 0.01% water). The organic solvent can bepresent in the solution in an amount that is the remaining balance afterother components have been added to the fixative solution. The organicsolvent can serve as a drying agent for a specimen and can displacewater from the specimen.

In some embodiments, the fixative solution includes a cytological dye.The dye can increase the staining of a specimen and can include, forexample, Azure B, methylene blue, eosin, thiazine stains, andfluorescein derivatives. The dye can be at least about 80% pure (e.g.,at least about 85% pure, at least about 90% pure, at least about 95%pure, or at least about 99% pure). A representative HPLC chromatogram ofazure B is shown, for example, in FIG. 1. The dye can have aconcentration of from approximately 0.5 g/L (e.g., approximately 0.75g/L, approximately one g/L, approximately two g/L, approximately threeg/L, or approximately four g/L) to approximately five g/L (e.g.,approximately four g/L, approximately three g/L, approximately two g/L,approximately one g/L, or approximately 0.75 g/L) in the fixativesolution. For example, the fixative solution can include approximatelyone g/L of Azure B. In some embodiments, the fixative solution caninclude approximately 0.8 g/L of Azure B.

The fixative solution can include a surfactant. Without wishing to bebound by any theory, it is believed that a surfactant reduces thesurface tension of a solvent, and provides good spreadability of asolution onto a sample substrate at separation 92 as shown in FIG. 13B.In some embodiments, the surfactant is non-ionic, and can minimize thelikelihood of precipitation from solution that can result from ionicinteractions with components within a formulation, such as an ionicallycharged dye. A solution having little or no precipitates can be moreeasily discharged from a dispensing nozzle and can reduce the likelihoodthat the dispensing nozzle will clog or that a liquid flow is diminishedover time. In some embodiments, the surfactant can decrease thelikelihood of non-specific binding of components within a fixativesolution to the specimen, which can minimize imaging artifacts.

The fixative solution can include from approximately 0.05% (e.g., fromapproximately 0.075%, from approximately 0.1%, from approximately 0.2%,from approximately 0.3%, or from approximately 0.4%) to approximately0.5% (e.g., to approximately 0.4%, to approximately 0.3%, toapproximately 0.2%, to approximately 0.1%, or to approximately 0.075%)by volume of a liquid surfactant or by weight of a solid surfactant. Insome embodiments, the fixative solution can include from approximately0.5 to approximately two mL/L of a surfactant (e.g., a non-ionicsurfactant). For example, the fixative solution can includeapproximately one mL/L of polysorbate 20 (e.g., Tween 20).

The surfactant may be non-ionic, cationic, anionic or zwitterionic.Mixtures of surfactants may also be used. Exemplary classes ofsurfactants include alcohol ether sulfates, alcohol sulfates,alkanolamides, alkyl sulfonates, amine oxides, amphoteric surfactants,anionic surfactants, betaine derivatives, cationic surfactants,disulfonates, dodecylbenzene, sulfonic acid, ethoxylated alcohols,ethoxylated alkyl phenols, ethoxylated fatty acids, glycerol estershydrotropes, lauryl sulfates, mono and diglycerides, non-ionicsurfactants, phosphate esters, quaternary surfactants, and sorbitanderivatives.

Examplary non-ionic surfactants include, for example,BigCHAP(N,N-Bis[3-(D-gluconamido)propyl]cholamide), Bis(polyethyleneglycol bis[imidazoyl carbonyl]), Brij® 30 (Polyoxyethylene 4 laurylether) Brij® 35 (Polyoxyethylene 23 lauryl ether), Brij® 52(Polyoxyethylene 2 cetyl ether), Brij® 56 (Polyoxyethylene 10 cetylether), Brij® 58 (Polyoxyethylene 20 cetyl ether), Brij® 72(Polyoxyethylene 2 stearyl ether), Brij® 76 (Polyoxyethylene 10 stearylether), Brij® 78 (Polyoxyethylene 20 stearyl ether), Brij® 92(Polyoxyethylene 2 oleyl ether), Brij® 97 (Polyoxyethylene 10 oleylether), Brij® 98 (Polyoxyethylene 20 oleyl ether), Brij® 700(Polyoxyethylene 100 stearyl ether), Cremophor® EL (castor oil/ethyleneoxide polyether), Decaethylene glycol monododecyl ether,octanoyl-N-methylglucamide (MECA-8), decanoyl-N-methylglucamide(MECA-10), n-octylglucoside, n-dodecylglucoside,isotridecyl-poly(ethyleneglycolether)_(n), N-Decanoyl-N-methylglucamine,n-Decyl α-Dglucopyranoside, Decyl β-D-maltopyranoside,n-Dodecanoyl-N-methylglucamide, n-Dodecyl α-D-maltoside, n-Dodecylβ-D-maltoside, Heptaethylene glycol monodecyl ether, Heptaethyleneglycol monotetradecyl ether, n-Hexadecyl β-D-maltoside, Hexaethyleneglycol monododecyl ether, Hexaethylene glycol monohexadecyl ether,Hexaethylene glycol monooctadecyl ether, Hexaethylene glycolmonotetradecyl ether, Igepal CA-630 (Octylphenyl-polyethylene glycol),Igepal® CA-210 (polyoxyethylene(2) isooctylphenyl ether), Igepal® CA-520(polyoxyethylene(5) isooctylphenyl ether), Igepal® CO-630(polyoxyethylene(9)nonylphenyl ether), Igepal® CO-720(polyoxyethylene(12) nonylphenyl ether), Igepal® CO-890(polyoxyethylene(40) nonylphenyl ether), Igepal® CO-990(polyoxyethylene(100) nonylphenyl ether), Igepal® DM-970(polyoxyethylene(150) dinonylphenyl ether), Methy1-6-O—(N-heptylcarbamoyl)-α-D-glucopyranoside, Nonaethylene glycolmonododecyl ether, N-Nonanoyl-N-methylglucamine, Octaethylene glycolmonodecyl ether, Octaethylene glycol monododecyl ether, Octaethyleneglycol monohexadecyl ether, Octaethylene glycol monooctadecyl ether,Octaethylene glycol monotetradecyl ether, Octyl-β-D-glucopyranoside,Pentaethylene glycol monodecyl ether, Pentaethylene glycol monododecylether, Pentaethylene glycol monohexadecyl ether, Pentaethylene glycolmonohexyl ether, Pentaethylene glycol monooctadecyl ether, Pentaethyleneglycol monooctyl ether, Polyethylene glycol diglycidyl ether,Polyethylene glycol ether W-1, Polyoxyethylene 10 tridecyl ether,Polyoxyethylene 100 stearate, Polyoxyethylene 20 isohexadecyl ether,Polyoxyethylene 20 oleyl ether, Polyoxyethylene 40 stearate,Polyoxyethylene 50 stearate, Polyoxyethylene 8 stearate, Polyoxyethylenebis(imidazolyl carbonyl), Polyoxyethylene 25 propylene glycol stearate,Saponin, Span® 20 (Sorbitan monolaurate), Span® 40 (Sorbitanmonopalmitate), Span® 60 (Sorbitan mono stearate), Span® 65 (Sorbitantristearate), Span® 80 (Sorbitan monooleate), Span® 85 (Sorbitantrioleate), Tergitol in any form (including Types15-S-5,15-S-7,15-S-9,15-S-12, 15-S-30, NP-4, NP-7, NP-9, NP-10, NP-40,NPX (Imbentin-N/63), TMN-3 (Polyethylene glycol trimethylnonyl ether),TMN-6 (Polyethylene glycol trimethylnonyl ether), TMN-10 (Polyethyleneglycol trimethylnonyl ether), MIN FOAM 1×, and MIN FOAM 2×),Tetradecyl-β-D-maltoside, Tetraethylene glycol monodecyl ether,Tetraethylene glycol monododecyl ether, Tetraethylene glycolmonotetradecyl ether, Triethylene glycol monodecyl ether, Triethyleneglycol monododecyl ether, Triethylene glycol monohexadecyl ether,Triethylene glycol monooctyl ether, Triethylene glycol monotetradecylether, Triton® CF-21, Triton® CF-32, Triton® DF-12, Triton® DF-16,Triton® GR-5M, Triton® N-101 (Polyoxyethylene branched nonylphenylether), Triton® QS-15, Triton® QS-44, Triton® RW-75 (Polyethylene glycol260 mono (hexadecyl/octadecyl)ether and 1-Octadecanol), Triton® X-100(Polyethylene glycol tert-octylphenyl ether), Triton® X-102, Triton®X-15, Triton® X-151, Triton® X-200, Triton® X-207, Triton® X-114,Triton® X-165, Triton® X-305, Triton® X-405 (polyoxyethylene (40)isooctylphenyl ether), Triton® X-405 reduced (polyoxyethylene (40)isooctylcyclohexyl ether), Triton® X-45 (Polyethylene glycol4-tert-octylphenyl ether), Triton® X-705-70, TWEEN® in any form(including TWEEN® 20 (Polyoxyethylenesorbitan monolaurate, orpolysorbate 20), TWEEN® 21 (Polyoxyethylenesorbitan monolaurate), TWEEN®40 (polyoxyethylene(20) sorbitan monopalmitate), TWEEN® 60 (Polyethyleneglycol sorbitan monostearate), TWEEN® 61 (Polyethylene glycol sorbitanmonostearate), TWEEN® 65 (Polyoxyethylenesorbitan Tristearate), TWEEN®80 (Polyoxyethylenesorbitan monooleate), TWEEN® 81(Polyoxyethylenesorbitan monooleate), and TWEEN® 85 (polyoxyethylene(20)sorbitan trioleate)), Tyloxapol (4-(1,1,3,3-tetramethylbutyl) phenolpolymer with formaldehyde and oxirane), and n-Undecylβ-D-glucopyranoside.

Exemplary anionic surfactants include Chenodeoxycholic acid, Cholicacid, Dehydrocholic acid, Deoxycholic acid, Digitonin, Digitoxigenin,N,N-Dimethyldodecylamine N-oxide, Sodium docusate, Sodiumglycochenodeoxycholate, Glycocholic acid, Glycodeoxycholic acid,Glycolithocholic acid 3-sulfate disodium salt, Glycolithocholic acidethyl ester, N-Lauroylsarcosine, Lithium dodecyl sulfate, Lugol (IodinePotassium Iodide), Niaproof (2-Ethylhexyl sulfate sodium salt), Niaproof4 (7-Ethyl-2-methyl-4-undecyl sulfate sodium salt), optionallysubstituted alkylsulfonate salts (including salts of 1-butanesulfonate,pentanesulfonate, hexanesulfonate, 1-Octanesulfonate, 1-decanesulfonate,1-dodecanesulfonate, 1-heptanesulfonate, 1-heptanesulfonate,1-nonanesulfonate, 1-propanesulfonate, and 2-bromoethanesulfonate,especially the sodium salts), Sodium cholate, Sodium deoxycholate,optionally substituted Sodium dodecyl sulfate, Sodium octyl sulfate,Sodium taurocholate, Sodium taurochenodeoxycholate, Sodiumtaurohyodeoxycholate, Taurolithocholic acid 3-sulfate disodium salt,Tauroursodeoxycholic acid sodium salt, Trizma® dodecyl sulfate,Ursodeoxycholic acid. The anionic surfactant can be provided in acid orsalt form, or both.

Exemplary cationic surfactants include Alkyltrimethylammonium bromide,Benzalkonium chloride, Benzyldimethylhexadecylammonium chloride,Benzyldimethyltetradecylammonium chloride, Benzyldodecyldimethylammoniumbromide, Benzyltrimethylammonium tetrachloroiodate,Dimethyldioctadecylammonium bromide, Dodecylethyldimethylammoniumbromide, Dodecyltrimethylammonium bromide,Ethylhexadecyldimethylammonium bromide, Girard's reagent T,Hexadecyltrimethylammonium bromide,N,N′,N′-Polyoxyethylene(10)-N-tallow-1,3-diaminopropane, Thonzoniumbromide, and Trimethyl(tetradecyl) ammonium bromide.

Exemplary zwitterionic surfactants include CHAPS(3-{(3-cholamidopropyl)-dimethylammonio}-1-propane-sulfonate), CHAPSO(3-{(3-cholamidopropyl)dimethyl-ammonio}-2-hydroxy-1-propane-sulfonate),3-(Decyldimethy lammonio) propanesulfonate, 3-(Dodecyldimethylammonio)propanesulfonate, 3-(N,N-Dimethylmyristylammonio) propanesulfonate,3-(N,N-Dimethyloctadecylammonio) propanesulfonate,3-(N,N-Dimethyloctylammonio) propanesulfonate, and3-(N,N-Dimethylpalmitylammonio) propanesulfonate.

The surfactant includes those known or discoverable in the art. Thesurfactant can be synthesized or can be obtained commercially from anyof a variety of vendors (e.g., Sigma Aldrich Corp., St. Louis, Mo., USA,www.sigmaaldrich.com). A given surfactant may be tested for its abilityto reduce artifacts by evaluating a sample treated with test surfactantagainst a sample processed without a surfactant treatment, and against asample processed with a surfactant or other positive control treatmentknown to reduce staining artifacts. A given surfactant may be tested forits ability to reduce precipitation of a formulation's components byvisually inspecting a solution containing a surfactant against asolution without surfactant, after the solutions have been allowed torest for at least two weeks.

The fixative solution can include an agent to minimize non-specificbinding of a dye to cellular components, such as ethylene glycol,propylene glycol, and/or polyethylene glycol. In some embodiments, thefixative solution can include from approximately 0.5% (e.g.,approximately 0.75%, approximately one %, approximately two %,approximately three %, or approximately four %) to approximately five %(e.g., approximately four %, approximately three %, approximately two %,approximately one %, or approximately 0.75%) by volume of a liquidnon-specific binding minimizing agent, or by weight of a solidnon-specific binding minimizing agent. For example, the fixativesolution can include from approximately five mL/L (e.g., approximately10 mL/L, approximately 15 mL/L, approximately 20 mL/L, approximately 25mL/L, approximately 30 mL/L, approximately 35 mL/L, approximately 40mL/L, or approximately 45 mL/L) to approximately 50 mL/L (e.g.,approximately 45 mL/L, approximately 40 mL/L, approximately 35 mL/L,approximately 30 mL/L, approximately 25 mL/L, approximately 20 mL/L,approximately 15 mL/L, or approximately 10 mL/L) ethylene glycol. Insome embodiments, the fixative solution includes approximately 10 mL/L(e.g., approximately 15 mL/L, approximately 20 mL/L, or approximately 25mL/L) ethylene glycol.

The fixative solution can include a buffering agent. Examples ofbuffering agents include HEPES buffer (e.g., a HEPES sodium salt and/ora HEPES free acid), MES, vis-tris, and other organic buffers. Thefixative solution can include from approximately 0.5 mM (e.g.,approximately one mM, approximately three mM, approximately five mM,approximately seven mM, or approximately nine mM) to approximately 10 mM(e.g., approximately nine mM, approximately seven mM, approximately fivemM, approximately three mM, or approximately one mM) of a bufferingagent. For example, the fixative solution can include approximately 1.5mM (e.g., approximately two mM or approximately one mM) HEPES sodiumsalt. The fixative solution can include from approximately 0.1 g/L toapproximately 10 g/L HEPES (e.g., from approximately 0.5 g/L toapproximately 10 g/L, from approximately 0.25 g/L to approximately 0.38,approximately 0.32 g/L).

The fixative solution can have a pH of from approximately 6 toapproximately 8 (e.g., a pH of from approximately 6.3 to approximately7.7, a pH of from approximately 6.5 to approximately 7.5, a pH of fromapproximately 6.7 to approximately 7.3, a pH of from approximately 6.8to approximately 7.2, a pH of from approximately 6.9 to approximately7.1, or a pH of approximately 7.0) when diluted in water at a ratio ofabout 1:10 fixative solution to water. In some embodiments, the fixativecan have an absorbance of approximately from 0.1 to one (e.g.,approximately from 0.1 to 0.8, approximately from 0.1 to 0.7,approximately from 0.1 to 0.5, approximately from 0.1 to 0.4,approximately from 0.1 to 0.3, approximately from 0.15 to 0.3,approximately from 0.15 to 0.2, approximately from 0.185 to 0.205,approximately 0.19, or approximately 0.2) at a peak wavelength of fromabout 640 to about 650 nm (e.g., about 646 to about 648 nm) at adilution of 1:1000 fixative solution to water.

As an example, to make the fixative solution, an organic solvent, suchas methanol, can first be added to a mixing vessel to less than 100%(e.g., approximately 90%) of the final desired volume. Calculatedamounts of a non-specific binding minimizer agent (e.g., ethyleneglycol), a surfactant (e.g., polysorbate 20), a cytological dye (e.g.,Azure B), and a buffering agent (e.g., HEPES sodium salt) can be addedto the methanol. Further methanol can be added to bring solution to itsfinal desired volume. The mixture can be mixed with a magnetic stirplate/stir bar and/or an impeller for a minimum of about 30 minutes.After mixing, a 1:10 dilution of the fixative solution in distilledwater can be prepared, and a pH reading can be carried out using a pHmeter (e.g., a Mettler pH meter). In some embodiments, if the pH is notwithin a desired range, then further buffering agent can be added to theundiluted fixative solution until a 1:10 dilution of the fixativesolution in distilled water reaches the desired pH.

Fixative solution absorbance can be measured using aUV-spectrophotometer (e.g., a Hitachi UV-spectrophotometer). A baselinecan first be run on the spectrophotometer. For example, a 10 mL sampleof the fixative solution can be filtered through a 0.45 μm syringefilter, the solution can be diluted 1:1000 with distilled water and runon the spectrophotometer at between approximately 500 to approximately700 nm. The absorbance at about 640 to about 650 nm can be recorded. Arepresentative UV-Vis absorption spectrum of a fixative solutionincluding azure B is shown, for example, in FIG. 2. If necessary,additional cytological dye can be added to the fixative solution tobring the absorbance to a desired range. If additional cytological dyeis added to the fixative solution, the solution is mixed for a minimumof about 30 minutes, and the measurement process (e.g., filtering a 10mL aliquot, diluting the aliquot in distilled water at a ratio of1:1000, and taking an absorbance measurement) is repeated. The pH of thesolution can also be re-measured and adjusted (if necessary) by themethod described above.

Finally, the fixative solution may be filtered through a 0.45 μm filterto remove any particulates before bottling. In some embodiments, a fineror coarser filter can be used. For example, a 0.1 to one μm filter(e.g., a 0.2 μm filter, a 0.4 μm filter, or a 0.8 μm filter) can be usedto remove any microorganisms and/or particulates in the fixativesolution. In some embodiments, the fixative can be stored in a 500 mLbottle and filled to 396 g±1 g, as measured by a balance. The pH of thefinal product can be measured, if desired. In some embodiments, afixative solution's HPLC chromatogram can be obtained, for example, toassess solution purity.

In some embodiments, the fixative solution includes a HEPES buffer(e.g., HEPES sodium salt and/or HEPES free acid) as it is soluble inmethanol, is compatible with Azure B, and/or can adjust pH to about 7.8.Azure B can preferentially stain certain cellular components (e.g., acell nucleus, basophils, cytoplasm, granules, etc.) to provide enhancedcontrast. Polysorbate 20 can enhance spreadability of the fixativesolution across a substrate (e.g., a microscope slide), and/or can primea nozzle for delivery of subsequent aqueous solutions. Ethylene glycolcan decrease non-specific binding of a dye to cell components (e.g.,chromatin to provide lighter colored nucleoli compared to a nucleus) soas to provide better contrast in a stained specimen.

For example, the cytological fixative solution can include about 0.5 g/Lto about 5.0 g/L Azure B; about 0.5 mL/L to about 2.0 mL/L polysorbate20; about 5 mL/L to about 50 mL/L ethylene glycol, propylene glycol, orpolypropylene glycol; about 0.1 g/L to about 10 g/L HEPES sodium salt;and methanol.

As another example, the cytological fixative solution can include about0.8 g/L to about 1.2 g/L Azure B; about 0.8 mL/L to about 1.2 mL/Lpolysorbate 20; about 9 mL/L to about 11 mL/L ethylene glycol; about0.25 g/L to about 0.38 g/L HEPES sodium salt; and methanol.

For example, the cytological fixative solution can include about one g/LAzure B; about one mL/L polysorbate 20; about 10 mL/L ethylene glycol;about 0.32 g/L HEPES sodium salt; and methanol.

As another example, the cytological fixative solution can include aboutone g/L Azure B; about 0.5 mL/L polysorbate 20; about 10 mL/L propyleneglycol; about 0.32 g/L HEPES sodium salt; and methanol.

As a further example, the cytological fixative solution can includeabout one g/L Azure B; about 1.5 mL/L polysorbate 20; about 10 mL/Lpolypropylene glycol; about 0.32 g/L HEPES sodium salt; and methanol.

First and Second Staining Solutions

Generally, the first staining solution can be an aqueous solution. Thesolvent can include distilled water or deionized water. The firststaining solution includes a cytological dye. The first stainingsolution can provide a red stain. The dye can increase the staining of aspecimen and can include, for example, eosin Y, and fluoresceinderivatives. In some embodiments, eosin Y can stain the cytoplasm andthe nuclei of cells within a specimen prepared, for example, from ablood sample. In some embodiments, eosinophils and neutrophils arepreferentially stained with eosin Y. The dye can be at least about 80%pure (e.g., at least about 85% pure, at least about 90% pure, at leastabout 95% pure, or about 100% pure). A representative HPLC chromatogramof Eosin Y is shown, for example, in FIG. 3. The dye can have aconcentration of from approximately 0.5 g/L (e.g., approximately 0.75g/L, approximately one g/L, approximately two g/L, approximately threeg/L, or approximately four g/L) to approximately five g/L (e.g.,approximately four g/L, approximately three g/L, approximately two g/L,approximately one g/L, or approximately 0.75 g/L) in the first stainingsolution. For example, the first staining solution can includeapproximately 0.75 g/L (e.g., approximately one g/L, approximately twog/L, approximately three g/L, approximately four g/L, or approximatelyfive g/L) eosin Y.

Generally, the second staining solution can be an aqueous solution. Thesolvent can include distilled water or deionized water. The secondstaining solution can include two or more cytological dyes. The secondstaining solution can provide a blue stain. The dyes can increase thestaining of a specimen and can include, for example, at least two ofazure B, methylene blue, and/or other thiazine dyes. In someembodiments, azure B can preferentially stain a cell nucleus, whilemethylene blue can primarily stain the cytoplasm and to a minor amount,the nucleus of cells in a specimen prepared, for example, from a bloodsample. The dyes can each independently be at least about 80% pure(e.g., at least about 85% pure, at least about 90% pure, at least about95% pure, or about 100% pure). Each dye can independently have aconcentration of from approximately 0.25 g/L (e.g., approximately 0.3g/L, approximately 0.4 g/L, approximately 0.45 g/L, approximately 0.5g/L, approximately 0.75 g/L, approximately one g/L, approximately 1.5g/L, or approximately two g/L) to approximately 2.5 g/L (e.g.,approximately two g/L, approximately 1.5 g/L, approximately one g/L,approximately 0.75 g/L, or approximately 0.5 g/L, approximately 0.45g/L, approximately 0.4 g/L, or approximately 0.3 g/L) in the secondstaining solution. For example, the second staining solution can includeapproximately 0.5 g/L azure B and approximately 0.45 g/L methylene bluehydrate. In some embodiments, the second staining solution can includeapproximately 1:4 to approximately 4:1 (e.g., approximately 3:1,approximately 2:1, approximately 1:1, approximately 1:2, orapproximately 1:3) ratio of two dyes, as assessed by the area under thelargest HPLC peak for each of the dyes when the absorbance is monitoredat a wavelength corresponding to the maximum absorbance for each dye(e.g., at about 630 nm and about 660 nm). A representative HPLCchromatograph of a second staining solution including an approximately1:1 ratio of azure B to methylene blue is shown, for example, in FIG. 5.

The first and second staining solutions can each independently include asurfactant. Without wishing to be bound by any theory, it is believedthat a surfactant reduces the surface tension of a solvent, and canprovide good spreadability of a solution onto a sample substrate. Insome embodiments, the surfactant is non-ionic, and can minimize thelikelihood of precipitation from solution that can result, for example,from ionic interactions with components within a formulation, such as anionically charged dye. A solution having little or no precipitates canbe more easily discharged from a dispensing nozzle and can reduce thelikelihood that the dispensing nozzle will clog or that a liquid flow isdiminished. In some embodiments, the surfactant can decrease thelikelihood of non-specific binding or minimize artifacts that can occuras a result of non-specific binding. Solutions having little or noprecipitates can also minimize artifacts that can be present in asample.

The first and second staining solutions can each independently includefrom approximately 0.05% (e.g., approximately 0.075%, approximately0.1%, approximately 0.2%, approximately 0.3%, or approximately 0.4%) toapproximately 0.5% (e.g., approximately 0.4%, approximately 0.3%,approximately 0.2%, approximately 0.1%, or approximately 0.075%) byvolume of a liquid surfactant or by weight of a solid surfactant. Insome embodiments, the first and second staining solutions can eachindependently include from approximately 0.5 to approximately two mL/Lof a surfactant (e.g., a non-ionic surfactant). For example, the firstand second staining solutions can each independently includeapproximately one ml/L of polysorbate 20 (e.g., Tween 20).

The surfactant may be non-ionic, cationic, anionic or zwitterionic.Mixtures of surfactants may also be used. Exemplary classes ofsurfactants include alcohol ether sulfates, alcohol sulfates,alkanolamides, alkyl sulfonates, amine oxides, amphoteric surfactants,anionic surfactants, betaine derivatives, cationic surfactants,disulfonates, dodecylbenzene, sulfonic acid, ethoxylated alcohols,ethoxylated alkyl phenols, ethoxylated fatty acids, glycerol estershydrotropes, lauryl sulfates, mono and diglycerides, non-ionicsurfactants, phosphate esters, quaternary surfactants, and sorbitanderivatives. Exemplary surfactants are as previously provided under thesection entitled “Fixative Solution.”

The first and second staining solutions can each independently includean agent to minimize non-specific binding of a dye to cellularcomponents, such as ethylene glycol, propylene glycol, polyethyleneglycol. In some embodiments, the first and second staining solutions caneach independently include from approximately 0.5% (e.g., approximately0.75%, approximately one %, approximately two %, approximately three %,or approximately four %) to approximately five % (e.g., approximatelyfour %, approximately three %, approximately two %, approximately one %,or approximately 0.75%) by volume of a liquid non-specific bindingminimizing agent, or by weight of a solid non-specific bindingminimizing agent. For example, the first and second staining solutionscan each independently include from approximately five mL/L (e.g.,approximately 10 mL/L, approximately 15 mL/L, approximately 20 mL/L,approximately 25 mL/L, approximately 30 mL/L, approximately 35 mL/L,approximately 40 mL/L, or approximately 45 mL/L) to approximately 50mL/L (e.g., approximately 45 mL/L, approximately 40 mL/L, approximately35 mL/L, approximately 30 mL/L, approximately 25 mL/L, approximately 20mL/L, approximately 15 mL/L, or approximately 10 mL/L) ethylene glycol.In some embodiments, the first and second staining solutions can eachindependently include approximately 10 mL/L (e.g., approximately 15mL/L, approximately 20 mL/L, or approximately 25 mL/L) ethylene glycol.

The first and second staining solutions can each independently include abuffering agent. Examples of buffering agents include bis-tris buffer,phosphate, HEPES, MES, Tris, and organic buffers having a pH betweenabout 5 and about 8. The first and second staining solutions can eachindependently include from approximately five mM (e.g., approximately 25mM, approximately 50 mM, approximately 100 mM, approximately 150 mM, orapproximately 200 mM) to approximately 250 mM (e.g., approximately 200mM, approximately 150 mM, approximately 100 mM, approximately 50 mM, orapproximately 25 mM) of a buffering agent. For example, the first andsecond staining solutions can each independently include approximately50 mM bis-tris. In some embodiments, the buffering agent in the secondstain solution is the same as the buffering agent in the first stainsolution, to increase compatibility between the stain solutions.

The first and second staining solutions can each independently include asalt, such as sodium chloride, potassium chloride, sodium acetate,calcium chloride, magnesium chloride, sodium sulfate, magnesium sulfate,and ammonium sulfate. The first and second staining solutions can eachindependently include a salt concentration of from approximately one g/L(e.g., approximately two g/L, approximately five g/L, approximately 10g/L, or approximately 15 g/L) to approximately 20 g/L (e.g.,approximately 15 g/L, approximately 10 g/L, approximately five g/L, orapproximately two g/L). For example, the first and second stainingsolutions can each independently include approximately four g/L(approximately three g/L or approximately two g/L) of a salt, such assodium chloride. For example, the second staining solution can includeapproximately two g/L (e.g., approximately three g/L, or approximatelyfour g/L) of a salt, such as sodium chloride. A salt can diminishnon-specific binding of a cytological dye to a specimen. Without wishingto be bound by any theory, it is believed that the negative and positiveions of a salt can shield non-specific charge-charge interactionsbetween a dye and a specimen by, for example, binding to charged specieswithin a specimen.

The first and second staining solution can each independently include anantimicrobial agent. An antimicrobial agent can inhibit the growth ofmicroorganisms and increase the shelf life of a staining solution. Theantimicrobial agent can include benzalkonium chloride,5-chloro-2-methyl-4-isothiazolin-3-one, 2-methyl-4-isothiazolin-3-one,ProClin® (e.g., ProClin 300®), azides, merthiolates, and/or antibiotics.In some embodiments, the antimicrobial agent includes5-chloro-2-methyl-4-isothiazolin-3-one and2-methyl-4-isothiazolin-3-one. For example, the antimicrobial agent canbe ProClin® 300, which can contain about 2.3%5-chloro-2-methyl-4-isothiazolin-3-one and about 0.7%2-methyl-4-isothiazolin-3-one in inert solvents (e.g., modified glycoland alkyl carboxylate) available from Sigma-Aldrich. The first andsecond stain solutions can each independently include the antimicrobialagent at a concentration of from approximately 0.2 ppm (e.g.,approximately one ppm, approximately five ppm, approximately 10 ppm,approximately 20 ppm, approximately 30 ppm, or approximately 40 ppm) toapproximately 50 ppm (e.g. approximately 40 ppm, approximately 30 ppm,approximately 20 ppm, approximately 10 ppm, approximately five ppm, orapproximately one ppm). In some embodiments, the first and secondstaining solutions can each independently contain approximately 15 ppm(approximately 10 ppm, approximately 5 ppm, or approximately 2 ppm)ProClin 300®.

In some embodiments, the first and second staining solutions can eachindependently further include an acid to adjust a pH. The acid can beany acid traditionally used to adjust the pH of a solution. For example,acetic acid, nitric acid, hydrochloric acid, phosphoric acid, formicacid, sulfuric acid, or citric acid can be used.

The first and second staining solutions can each independently have a pHof from approximately 5 to approximately 8 (e.g., from approximately 5.5to approximately 8, from approximately 5.5 to approximately 7.5, fromapproximately 5.5 to approximately 7, from approximately 5.5 toapproximately 6, from approximately 5.8 to approximately 6.2, fromapproximately 5.9 to approximately 6.1, a pH of approximately 6, or a pHof approximately 7). In some embodiments, the first staining solutioncan have an absorbance of from approximately 0.1 to approximately 1(e.g., from approximately 0.1 to approximately 0.8, from approximately0.1 to approximately 0.7, from approximately 0.1 to approximately 0.5,from approximately 0.1 to approximately 0.4, from approximately 0.1 toapproximately 0.3, from approximately 0.15 to approximately 0.3, fromapproximately 0.15 to approximately 0.2, from approximately 0.185 toapproximately 0.205, approximately 0.19, or approximately 0.2) at amaximum peak wavelength of from about 510 to about 530 nm (e.g., about510 to about 520 nm, or about 515 to about 517 nm) at a dilution inwater of 1:500 first staining solution to water. In some embodiments,the second staining solution can have an absorbance of fromapproximately 0.1 to approximately 1 (e.g., from approximately 0.1 toapproximately 0.8, from approximately 0.1 to approximately 0.7, fromapproximately 0.1 to approximately 0.5, from approximately 0.1 toapproximately 0.4, from approximately 0.1 to approximately 0.3, fromapproximately 0.15 to approximately 0.3, from approximately 0.15 toapproximately 0.2, from approximately 0.185 to approximately 0.205,approximately 0.19, or approximately 0.2) at a peak wavelength of fromabout 630 to about 660 nm (e.g., about 640 to about 660 nm, about 640 toabout 655 nm, about 645 to about 655 nm, about 650 to about 655 nm, orabout 650.5 to about 652.5 nm) at a dilution of 1:1000 second stainingsolution to water.

In some embodiments, to make the first or second staining solution,distilled or deionized water is first added to a mixing vessel to lessthan 100% (e.g., approximately 90%) of the final desired volume.Calculated amounts of a non-specific binding minimizer agent (e.g.,ethylene glycol), a surfactant (e.g., polysorbate 20), one or morecytological dyes (e.g., eosin Y, or azure B and methylene blue), a salt(e.g., NaCl), an antimicrobial agent (e.g., ProClin 300®), an acid(e.g., acetic acid), and/or a buffering agent (e.g., bis tris) can beadded to the water. Further water can be added to bring the solution toits final desired volume. The mixture can be mixed with a magnetic stirplate/stir bar and/or an impeller for a minimum of about 30 minutes.After mixing, a pH reading is carried out on an aliquot of the first orsecond staining solution using a pH meter (e.g., a Mettler pH meter). Insome embodiments, if the pH is not within a desired range (e.g., five toeight), then further acid can be added to the first or second stainingsolution until the desired pH is attained.

First staining solution absorbance can be measured using aUV-spectrophotometer (e.g., a Hitachi UV-spectrophotometer). A baselinecan first be run on the spectrophotometer. For example, a 10 mL sampleof the first staining solution can be filtered through a 0.45 μm syringefilter, diluted 1:500 with distilled water, and then scanned on thespectrophotometer at between 500-700 nm. The absorbance at 510 to 530 nmcan be recorded. A representative UV-Vis absorption spectrum of a firststaining solution including eosin Y is shown, for example, in FIG. 4. Ifnecessary, additional cytological dye can be added to the first stainingsolution to bring the absorbance to a desired range. If additionalcytological dye (e.g., eosin) is added to the first staining solution,the solution can be mixed for a minimum of about 30 minutes, and themeasurement process (e.g., filtering a 10 mL aliquot, diluting thealiquot in distilled water at a ratio of 1:500, and taking an absorbancemeasurement) can be repeated. The pH of the solution can be re-measuredand adjusted to pH 5-8 (if necessary) by the method above.

Second staining solution absorbance can be measured using aUV-spectrophotometer (e.g., a Hitachi UV-spectrophotometer). A baselinecan first be run on the spectrophotometer. A 10 mL sample of the secondstaining solution can be filtered through a 0.45 μm syringe filter,diluted 1:1000 with distilled water, and then scanned on thespectrophotometer at between 500-700 nm. The absorbance at 630-660 nmcan be recorded. A representative UV-Vis absorption spectrum of a secondstaining solution including 1:1 methylene blue to azure B is shown, forexample, in FIG. 6. If necessary, additional cytological dye can beadded to the second staining solution to bring the absorbance to adesired range. A portion of the filtered sample can also bechromatographed using high-performance liquid chromatography (HPLC) andthe area of the tallest absorbance peak corresponding to each dye withinthe sample can be recorded. The peak areas can be approximatelyequivalent between the different dye constituents.

If the HPLC peak areas for the dyes within the second staining solutionare not approximately equivalent, additional cytological dye (e.g.,azure B and/or methylene blue) can be added to the second stainingsolution until the solution can include approximately equal amounts (asassessed by the peak areas) of each of the dyes. After adding one ormore dyes, the second staining solution can be mixed for a minimum ofabout 30 minutes, and the measurement process (e.g., filtering a 10 mLaliquot, diluting the aliquot in distilled water at a ratio of 1:500,and taking absorbance and HPLC measurements) can be repeated. The pH ofthe solution can be re-measured and adjusted to a desired range (ifnecessary) by the method described above.

Finally, the first and second staining solution can each beindependently filtered through a 0.45 μm filter to remove anyparticulates before bottling. In some embodiments, a finer or coarserfilter can be used. For example, a 0.1 to one μm filter (e.g., a 0.2 μmfilter, a 0.4 μm filter, or a 0.8 μm filter) can be used to remove anymicroorganisms and/or particulates in the first or second stainingsolution. In some embodiments, the first or second staining solution caneach be independently stored in a 250 mL bottle and filled to 250 g±1 g,as measured by a balance. The pH of the final product can be measured,if desired. In some embodiments, a first staining solution's HPLCchromatogram can be obtained, for example, to assess solution purity. Insome embodiments, a second staining solution's HPLC chromatogram can beobtained, for example, to assess solution purity and/or to obtain aratio of the dyes within the solution.

In some embodiments, a first stain solution can stain certain cells of aspecimen prepared from, for example, a blood sample (e.g., eosinophils,neutrophils). The first stain solution can contain a bis-tris buffer,and the resulting stain solution can provide a redder color and a morevisually pleasing stained specimen. The bis-tris buffer can becompatible with eosin Y. In some embodiments, azure B is included inmore than one formulation, such as in a fixative solution and a secondstain solution. When a specimen is exposed more than once to azure B, itis believed that better staining of the specimen can occur compared to asingle exposure to azure B.

The first and second stain solutions can each independently contain NaCland ethylene glycol, which together can provide a synergistic shieldingeffect to non-specific binding, i.e., where a formulation containingboth NaCl and ethylene glycol can have less non-specific binding than aformulation containing an equivalent concentration of either NaCl orethylene glycol. For example, a formulation including both NaCl andethylene glycol can provide a lighter colored nucleoli than theremaining nucleus, while a formulation without NaCl and ethylene glycolcan provide a uniformly dark nucleus. In some embodiments, a stainsolution including NaCl can have decreased background staining comparedto a stain solution without NaCl.

For example, the first cytological staining solution can include about0.5 g/L to about 5.0 g/L Eosin Y; about 5 mM to about 250 mM bis-tris orphosphate buffer; about 0.5 mL/L to about 2.0 mL/L polysorbate 20; aboutone g/L to about 20 g/L sodium chloride; about five mL/L to about 50mL/L ethylene glycol; about 0.2 ppm to about 50 ppm ProClin 300®; aceticacid; and water, wherein the solution has a pH of from about 5.8 toabout 6.2.

As another example, the first cytological staining solution can includeabout 0.6 g/L to about 0.9 g/L Eosin Y; about 45 mM to about 55 mMbis-tris buffer; about 0.8 mL/L to about 1.2 mL/L polysorbate 20; aboutthree g/L to about five g/L sodium chloride; about nine mL/L to about 11mL/L ethylene glycol; about 10 ppm to about 20 ppm ProClin 300®; aceticacid; and water, wherein the solution has a pH of from about 5.8 toabout 6.2.

For example, the first cytological staining solution can include about0.75 g/L Eosin Y; about 50 mM bis-tris buffer; about one mL/Lpolysorbate 20; about four g/L sodium chloride; about 10 mL/L ethyleneglycol; about 15 ppm ProClin 300®; acetic acid; and water, wherein thesolution has a pH of from about 5.8 to about 6.2.

As another example, the first cytological staining solution can includeabout 0.75 g/L Eosin Y; about 50 mM bis-tris buffer; about 0.5 mL/Lpolysorbate 20; about six g/L sodium chloride; about 20 mL/L ethyleneglycol; about 15 ppm ProClin 300®; acetic acid; and water, wherein thesolution has a pH of from about 5.8 to about 6.2.

As a further example, the first cytological staining solution caninclude about 0.75 g/L Eosin Y; about 50 mM phosphate buffer; about 2.0mL/L polysorbate 20; about four g/L sodium chloride; about 50 mL/Lethylene glycol; about 15 ppm ProClin 300®; acetic acid; and water,wherein the solution has a pH of from about 5.8 to about 6.2.

For example, the second cytological staining solution can include about0.25 to about 2.5 g/L Azure B; about 0.25 g/L to about 0.5 g/L methyleneblue; about 5 mM to about 250 mM bis-tris or HEPES buffer; about 0.5mL/L to about 2.0 mL/L polysorbate 20, about one g/L to about 20 g/Lsodium chloride, and about 0.2 ppm to about 50 ppm ProClin 300®; aceticacid; and water, wherein the solution has a pH of from about 6.8 toabout 7.2.

As another example, the second cytological staining solution can includeabout 0.4 to about 0.6 g/L Azure B; about 0.4 g/L to about 0.5 g/Lmethylene blue; about 45 mM to about 55 mM bis-tris buffer; about 0.8mL/L to about 1.2 mL/L polysorbate 20, about 1.8 g/L to about 2.2 g/Lsodium chloride, and about 10 ppm to about 20 ppm ProClin 300®; aceticacid; and water, wherein the solution has a pH of from about 6.8 toabout 7.2.

For example, the second cytological staining solution can include about0.5 g/L Azure B; about 0.45 g/L methylene blue; about 50 mM bis-trisbuffer; about one mL/L polysorbate 20, about two g/L sodium chloride,and about 15 ppm ProClin 300®; acetic acid; and water, wherein thesolution has a pH of from about 6.8 to about 7.2.

As another example, the second cytological staining solution can includeabout 0.5 g/L Azure B; about 0.45 g/L methylene blue; about 50 mM HEPESbuffer; about 0.5 mL/L polysorbate 20, about two g/L sodium chloride,and about 15 ppm ProClin 300®; acetic acid; and water, wherein thesolution has a pH of from about 6.8 to about 7.2.

As a further example, the second cytological staining solution caninclude about 0.5 g/L Azure B; about 0.45 g/L methylene blue; about 50mM HEPES buffer; about one mL/L polysorbate 20, about one g/L sodiumchloride, and about 15 ppm ProClin 300®; acetic acid; and water, whereinthe solution has a pH of from about 6.8 to about 7.2.

Rinse Solution

Generally, the rinse solution can be an aqueous solution. The solventcan include distilled water or deionized water. The rinse solution caninclude a non-specific binding minimizer agent, such as polyethyleneglycol, polyvinyl pyrrolidone, polyacrylic acid, polyvinyl alcohol,polysaccharides, and/or other hydrophilic water-soluble polymers. Insome embodiments, the non-specific binding minimizer agent can includeethylene glycol or propylene glycol. Without wishing to be bound by anytheory, it is believed that the non-specific binding minimizer agent canprepare a specimen for drying processes during sample preparation. Thenon-specific binding minimizer agent can provide a lacquer over thespecimen and can provide an enhanced visual appearance to the specimen.The non-specific binding minimizer agent can have a concentration offrom approximately one g/L (e.g., approximately two g/L, approximatelythree g/L, approximately four g/L, approximately five g/L, approximatelysix g/L, approximately seven g/L, approximately eight g/L, orapproximately nine g/L) to approximately ten g/L (e.g., approximatelynine g/L, approximately eight g/L, approximately seven g/L,approximately six g/L, approximately five g/L, approximately four g/L,approximately three g/L, or approximately two g/L) in the rinsesolution. For example, the rinse solution can include approximately fiveg/L polyethylene glycol (e.g., polyethylene glycol 1450).

The rinse solution can include a surfactant. Without wishing to be boundby any theory, it is believed that a surfactant reduces the surfacetension of a solvent, and can provide good spreadability of a solutiononto a sample substrate. In some embodiments, the surfactant isnon-ionic, and can minimize the likelihood of precipitation of solutioncomponents that can result, for example, from ionic interactions withcomponents within a formulation, such as an ionically charged dye. Asolution having little or no precipitates can be more easily dischargedfrom a dispensing nozzle and can reduce the likelihood that thedispensing nozzle will clog or that a liquid flow is diminished. In someembodiments, the surfactant can decrease the likelihood of non-specificbinding or minimize artifacts that can occur as a result of non-specificbinding. A solution having little or no precipitates can minimizeartifacts that can be present in a specimen.

The rinse solution can include from approximately 0.05% (e.g.,approximately 0.075%, approximately 0.1%, approximately 0.2%,approximately 0.3%, or approximately 0.4%) to approximately 0.5% (e.g.,approximately 0.4%, approximately 0.3%, approximately 0.2%,approximately 0.1%, or approximately 0.075%) by volume of a liquidsurfactant or by weight of a solid surfactant. In some embodiments, therinse solution can include from approximately 0.5 to approximately twomL/L of a surfactant (e.g., a non-ionic surfactant). For example, therinse solution can include approximately one ml/L of polysorbate 20(e.g., Tween 20).

The surfactant may be non-ionic, cationic, anionic or zwitterionic.Mixtures of surfactants may also be used. Exemplary classes ofsurfactants include alcohol ether sulfates, alcohol sulfates,alkanolamides, alkyl sulfonates, amine oxides, amphoteric surfactants,anionic surfactants, betaine derivatives, cationic surfactants,disulfonates, dodecylbenzene, sulfonic acid, ethoxylated alcohols,ethoxylated alkyl phenols, ethoxylated fatty acids, glycerol estershydrotropes, lauryl sulfates, mono and diglycerides, non-ionicsurfactants, phosphate esters, quaternary surfactants, and sorbitanderivatives. Exemplary surfactants are as previously provided under thesection entitled “Fixative Solution”.

The rinse solution can include a buffering agent. Examples of bufferingagents include HEPES buffer (e.g., HEPES sodium salt and/or HEPES freeacid), bis-tris buffer, phosphate, MES, Tris, and organic buffers havinga pH between 5 and 8. The rinse solution can include from approximatelyfive mM (e.g., approximately 25 mM, approximately 50 mM, approximately100 mM, approximately 150 mM, or approximately 200 mM) to approximately250 mM (e.g., approximately 200 mM, approximately 150 mM, approximately100 mM, approximately 50 mM, or approximately 25 mM) of a bufferingagent. For example, the rinse solution can include approximately 50 mMHEPES.

The rinse solution can include an antimicrobial agent. An antimicrobialagent can inhibit the growth of microorganisms and increase the shelflife of a rinse solution. The antimicrobial agent can includebenzalkonium chloride, 5-chloro-2-methyl-4-isothiazolin-3-one,2-methyl-4-isothiazolin-3-one, ProClin® (ProClin 300®), azides,merthiolates, and/or antibiotics. In some embodiments, the antimicrobialagent includes 5-chloro-2-methyl-4-isothiazolin-3-one and2-methyl-4-isothiazolin-3-one. For example, the antimicrobial agent canbe ProClin 300®, which can contain about 2.3%5-chloro-2-methyl-4-isothiazolin-3-one and about 0.7%2-methyl-4-isothiazolin-3-one in inert solvents (e.g., modified glycoland alkyl carboxylate) available from Sigma-Aldrich. The antimicrobialagent can be present at a concentration of from approximately 0.2 ppm(e.g., approximately one ppm, approximately five ppm, approximately 10ppm, approximately 20 ppm, approximately 30 ppm, or approximately 40ppm) to approximately 50 ppm (e.g. approximately 40 ppm, approximately30 ppm, approximately 20 ppm, approximately 10 ppm, approximately fiveppm, or approximately one ppm). In some embodiments, the rinse solutioncontains approximately 15 ppm (approximately 10 ppm, approximately 5ppm, or approximately 2 ppm) ProClin 300®.

In some embodiments, the rinse solution further includes an acid toadjust a pH. The acid can be any acid traditionally used to adjust thepH of a solution. For example, acetic acid, nitric acid, hydrochloricacid, phosphoric acid, formic acid, sulfuric acid, or citric acid can beused.

The rinse solution can include an alcohol, such as methanol, ethanol, orpropanol. The alcohol can remove excess dyes, can promote faster dryingof the rinse solution on the specimen and/or the substrate. In someembodiments, DMSO or other organic solvent in which the dyes are solublecan be used in the rinse solution and can provide complete fluidevacuation from a delivering nozzle. For example, the rinse solution caninclude from approximately 10 mL/L (e.g., approximately 25 mL/L,approximately 50 mL/L, approximately 75 mL/L, approximately 100 mL/L,approximately 125 mL/L, approximately 150 mL/L, or approximately 175mL/L) to approximately 200 mL/L (e.g., approximately 175 mL/L,approximately 150 mL/L, approximately 125 mL/L, approximately 100 mL/L,approximately 75 mL/L, approximately 50 mL/L, or approximately 25 mL/L)of an alcohol. In some embodiments, the rinse solution includesapproximately 50 mL/L methanol. In some embodiments, the rinse solutioncan have at most approximately 200 mL/L of an organic solvent, so that arinse solution cannot wash an excessive amount of desired dye from astained biological sample.

The rinse solution can have a pH of from approximately 5 toapproximately 8 (e.g., from approximately 5.5 to approximately 8, fromapproximately 5.5 to approximately 7.5, from approximately 5.5 toapproximately 7, from approximately 5.5 to approximately 6, fromapproximately 5.8 to approximately 6.2, from approximately 5.9 toapproximately 6.1, a pH of approximately 7.0, or a pH of approximately6.0). For example, the rinse solution can have a pH of approximately7.0.

To make the rinse solution, distilled or deionized water can be added toa mixing vessel to less than 100% (e.g., approximately 90%) of the finaldesired volume.

Calculated amounts of an alcohol (e.g., methanol), a non-specificbinding minimizer agent (e.g., PEG), a buffering agent (e.g., HEPESsodium salt and HEPES free acid), a surfactant (e.g., polysorbate 20),an acid (if used), and an antimicrobial agent (e.g., ProClin 300®) canbe added to the water. Further water can be added to bring the solutionto its final desired volume. The mixture can be mixed with a magneticstir plate/stir bar and/or an impeller for a minimum of about 30minutes. After mixing, a pH reading can be carried out on an aliquot ofthe rinse solution using a pH meter (e.g., a Mettler pH meter). In someembodiments, if the pH is not within a desired range (e.g., about fiveto about eight), then further acid can be added to the second stainingsolution until a desired pH is attained.

Finally, the rinse solution can be filtered through a 0.45 μm filter toremove any particulates before bottling. In some embodiments, a finer orcoarser filter can be used. For example, a 0.1 to one μm filter (e.g., a0.2 μm filter, a 0.4 μm filter, or a 0.8 μm filter) can be used toremove any microorganisms and/or particulates in the rinse solution. Insome embodiments, the rinse solution can be stored in a 500 mL bottleand filled to 500 g±1 g, as measured by a balance. The pH of the finalproduct can be measured, if desired.

In some embodiments, a rinse solution that includes polyethylene glycolthat is applied at the end of stain application can improve the visualappearance of a specimen, compared to a specimen treated with a rinsesolution without polyethylene glycol. In some embodiments, a rinsesolution including HEPES buffer at a pH of approximately 6.8 can improvethe appearance of cells, such as red blood cells, by providing a bettercolor balance between red and blue colors.

In some embodiments, the components of the rinse solution can be presentat concentrations lower than those described above (e.g., by a factor offive).

The polyethylene glycol component of the rinse solution can have aconcentration of from approximately 0.2 g/L (e.g., approximately 0.4g/L, approximately 0.6 g/L, approximately 0.8 g/L, approximately oneg/L, approximately 1.2 g/L, approximately 1.4 g/L, approximately 1.6g/L, or approximately 1.8 g/L) to approximately two g/L. For example,the rinse solution can include approximately one g/L polyethylene glycol(e.g., polyethylene glycol 1450).

The rinse solution can include from approximately 0.01% (e.g.,approximately 0.015%, approximately 0.02%, approximately 0.04%,approximately 0.06%, or approximately 0.8%) to approximately 0.1% byvolume of a liquid surfactant or by weight of a solid surfactant. Insome embodiments, the rinse solution can include from approximately0.0501% to 0.306% of a surfactant. In some embodiments, the rinsesolution can include from approximately 0.1 to approximately 0.4 mL/L ofa surfactant (e.g., a non-ionic surfactant). For example, the rinsesolution can include approximately 0.2 ml/L of polysorbate 20 (e.g.,Tween 20).

The rinse solution can include from approximately one mM (e.g.,approximately 5 mM, approximately 10 mM, approximately 20 mM,approximately 30 mM, or approximately 40 mM) to approximately 50 mM of abuffering agent. For example, the rinse solution can includeapproximately 10 mM HEPES.

The antimicrobial agent can be present at a concentration of fromapproximately 0.04 ppm (e.g., approximately 0.2 ppm, approximately oneppm, approximately two ppm, approximately five ppm, approximately sixppm, or approximately eight ppm) to approximately 10 ppm. In someembodiments, the rinse solution contains approximately three ppm(approximately two ppm, approximately one ppm, or approximately 0.4 ppm)ProClin 300®.

The rinse solution can include from approximately two mL/L (e.g.,approximately five mL/L, approximately ten mL/L, approximately 15 mL/L,approximately 20 mL/L, approximately 25 mL/L, approximately 30 mL/L, orapproximately 35 mL/L) to approximately 40 mL/L of an alcohol. In someembodiments, the rinse solution includes approximately ten mL/Lmethanol. In some embodiments, the rinse solution can have at mostapproximately 40 mL/L of an organic solvent, so that a rinse solutioncannot wash an excessive amount of desired dye from a stained biologicalsample.

For example, the rinse solution can include about 0.2 g/L to about teng/L polyethylene glycol; about 1 mM to about 250 mM HEPES, MES, orbis-tris buffer; about 0.1 mL/L to about 2.40 mL/L polysorbate 20; about0.04 ppm to about 50 ppm ProClin 300®; about 9 mL/L to about 200 mL/Lmethanol; and water, wherein the rinse solution has a pH of from about6.6 to about 7.0.

For example, the rinse solution can include about one g/L to about teng/L polyethylene glycol; about 5 mM to about 250 mM HEPES, MES, orbis-tris buffer; about 0.5 mL/L to about 2.0 mL/L polysorbate 20; about0.2 ppm to about 50 ppm ProClin 300®; about 10 mL/L to about 200 mL/Lmethanol; and water, wherein the rinse solution has a pH of from about6.6 to about 7.0.

As another example, the rinse solution can include about 4.5 g/L toabout 5.5 g/L polyethylene glycol; about 45 mM to about 55 mM HEPESbuffer; about 0.8 mL/L to about 1.2 mL/L polysorbate 20; about 10 ppm toabout 20 ppm ProClin 300®; about 45 mL/L to about 55 mL/L methanol; andwater, wherein the rinse solution has a pH of from about 6.6 to about7.0.

For example, the rinse solution includes about five g/L polyethyleneglycol; about 50 mM HEPES buffer; about one mL/L polysorbate 20; about15 ppm ProClin 300®; about 50 mL/L methanol; and water, wherein therinse solution has a pH of from about 6.6 to about 7.0.

As another example, the rinse solution includes about 10 g/Lpolyethylene glycol; about 50 mM MES buffer; about one mL/L polysorbate20; about 15 ppm ProClin 300®; about 50 mL/L methanol; and water,wherein the rinse solution has a pH of from about 6.6 to about 7.0.

As a further example, the rinse solution includes about 10 g/Lpolyethylene glycol; about 50 mM bis-tris buffer; about 0.5 mL/Lpolysorbate 20; about 15 ppm ProClin 300®; about 50 mL/L methanol; andwater, wherein the rinse solution has a pH of from about 6.6 to about7.0.

For example, the rinse solution can include about 0.2 g/L to about twog/L polyethylene glycol; about one mM to about 50 mM HEPES buffer; about0.16 mL/L to about 0.24 mL/L polysorbate 20; about 0.04 ppm to about 10ppm ProClin 300®; about 9 mL/L to about 11 mL/L methanol; and water,wherein the rinse solution has a pH of from about 6.6 to about 7.0.

As another example, the rinse solution can include about 0.9 g/L toabout 1.1 g/L polyethylene glycol; about 9 mM to about 11 mM HEPESbuffer; about 0.16 mL/L to about 0.24 mL/L polysorbate 20; about 2 ppmto about 10 ppm ProClin 300®; about 9 mL/L to about 11 mL/L methanol;and water, wherein the rinse solution has a pH of from about 6.6 toabout 7.0.

In still another example, the rinse solution includes about one g/Lpolyethylene glycol; about 10 mM HEPES buffer; about 0.2 mL/Lpolysorbate 20; about 3 ppm ProClin 300®; about 10 mL/L methanol; andwater, wherein the rinse solution has a pH of from about 6.6 to about7.0.

Kits

In some embodiments, a kit can include one or more bottles of each ofthe fixative solution, first stain solution, second stain solution,and/or a rinse solution. The kit can include instructions and labels.The labels can include, for example, lot information and expirationdate(s). FIG. 25 shows an example of a kit including a fixativesolution, first and second staining solutions, and a rinse solution.

In some embodiments, the processing steps include applying a fixativesolution, applying a fixative solution, applying a first stain solution,applying a second stain solution, applying a rinse solution, andapplying a rinse solution.

Specimen Preparation Systems and Methods

The fixative, staining, and rinse formulations above can be used in anapparatus or machine for preparing a specimen (as described, forexample, in U.S. application Ser. No. 13/293,050, filed Nov. 9, 2011,herein incorporated by reference in its entirety), to enhance the visualappearance of certain features in the specimens. FIG. 7 illustrates anembodiment of an apparatus or machine 1 for preparing a specimen on asubstrate 2 such as a microscope slide, cover slip, or other transparentsurface, for examination or imaging. Machine 1 can be incorporated intoan overall system for preparing and analyzing specimens comprising bodyfluids or other biological samples containing cells, such as system 2000shown in FIG. 21 and described below. Machine 1 can generally include,or form a portion of, a system that features a first station thatobtains a specimen, a second station that applies the specimen to asubstrate, third and fourth stations for fixing and staining thespecimen, respectively, a fifth station that dries the specimen, a sixthstation that images the specimen, and a seventh station for analyzingthe images and data obtained from the specimen. Certain embodiments ofmachine 1 are compatible with system 2000; some embodiments of machine 1can be used in other specimen preparation systems, and/or as stand-alonedevices.

In general, machine 1 may include one or more (e.g., two, three, four,five, or more than five) platforms 60A and 60B as shown in FIGS. 7-9 forspecimen processing. As shown in FIG. 8, platform 60A can includelateral sides for supporting a top side of the platform. A shield 100,shown in FIGS. 7 and 9, can be positioned between the platforms 60A and60B to prevent fluids from splattering between the platforms 60. In someembodiments, shield 100 can be formed from a transparent material thatblocks fluids from one of platforms 60A and 60B from contaminating theother platform. In certain embodiments, shield 100 can be formed from amaterial that is translucent or opaque. In FIGS. 7 and 9, shield 100 isdepicted as being formed from a transparent material to allow othercomponents positioned behind shield 100 to be shown in the same figure.Shield 100 could also have been shown as being formed from an opaquematerial, in which case portions of some components such as platform 60Aand block 80A would have been obscured.

FIG. 9B shows an indexing mechanism 50A that can be used to translatethe machine 1 to provide substrates 2 from each of the substrategrippers 20A, 20B to a position for specimen processing. The indexingmechanism 50A can be in many forms, such as electromechanical devices(e.g., a rack and pinion gear set powered by an electric motor), linearactuators (e.g., pneumatic actuators, hydraulic actuators, orelectromagnetic actuators). Although, in the illustrated embodiment, theindexing mechanism 50A translates the machine 1 linearly between twopositions, other translation paths are possible based on the number ofplatforms included on the machine 1, and their configuration and layout,such as circular or semi-circular (e.g., an indexing table that can movein an arcuate path). As shown, the indexing mechanism 50A can include agear rack 50B attached to a base 50C of the machine 1 and a pinion gear50D attached to an electric motor 50E that is fixed to the base 50C. Themachine 1 can be attached to the base 50C using one or more slidingdevices 50F so that the machine 1 can move smoothly when translated bythe indexing mechanism 50A. During use, the indexing mechanism 50A canmove the machine 1 so that the multiple substrate grippers 20A and/or20B of the machine 1 to receive a substrate 2 from a substrate mover 120(shown in FIG. 11) so that a sample disposed on the substrate 2 can beprepared by the machine 1, and also so that, once prepared, thesubstrate gripper 20A and/or 20B can provide the substrate 2 having aprepared sample can be provided to the substrate mover 120 for sampleprocessing.

For machines having two platforms 60A and 60B, as in the illustratedembodiment, substrates 2 are typically provided to, and from, thesubstrate mover 120 in an alternating manner. In some embodiments, afirst substrate 2 is provided from the substrate mover 120 to a firstsubstrate gripper 20A, to be processed at a first platform 60A, whilethe machine 1 is in a first position. While the first substrate 2 isprocessed at the first platform 60A, the indexing mechanism 50A cantranslate the machine 1 to a second position so that a second substrategripper 20B can receive a second substrate, to be processed at thesecond platform 60B, from the substrate mover 120. While the secondsubstrate is processed at the second platform 60B, the indexingmechanism 50A can translate the machine 1 back to the first position sothat the substrate mover 120 can remove the first substrate 2 from thefirst substrate gripper 20A. Once the substrate 2 is removed from thefirst gripping platform 20A, a next substrate can be provided to thefirst gripping platform 20A. This method for providing substrates toalternating gripping platforms can be implemented for more than two(e.g., three, four, five, or more than five) platforms therebyincreasing throughput of specimens prepared for further evaluation.

Platforms 60A and 60B are typically formed from one or more materialsthat are relatively chemically inert with respect to the fluids usedduring specimen processing and provide a suitable surface tension.Exemplary materials that can be used to form platforms 60A and 60Binclude engineering thermoplastics, such as polyoxymethylene (e.g.,Delrin® manufactured by DuPont), high molecular weight fluorocarbons,such as polytetrafluoroethylene (PTFE) (e.g., Teflon® manufactured byDuPont), and metals such as aluminum, steel, and titanium, provided theyare provided, manufactured, and/or treated to provide a suitable surfacetension that acts to assist in evenly distributing and confining theprocessing fluids to the space between substrate 2 and the platforms,and allowing suitable evacuation of the processing fluids as well. Byselection of suitable materials, the platforms can also advantageouslyreduce or minimize the formation of bubbles or spaces within the fluidsas they are distributed, and at the same time maintain a sufficientsurface tension such that fluid leakage out of the separation betweenthe platforms and substrate 2 is reduced or eliminated.

In general, the surface area of platforms 60A and 60B can be selected asdesired for purposes of substrate handling and fluid delivery. Factorssuch as the surface area of platforms 60A and 60B can also influence theselected surface area of substrate 2. For example, in some embodiments,the surface area of platform 60A (e.g., the area of the surface ofplatform 60A that faces substrate 2) is slightly smaller than the areaof the surface of substrate 2 that faces platform 60A. By maintainingsuch a relationship between the areas of the facing surfaces of platform60A and substrate 2, fluid leakage from the region between the surfacescan be reduced or eliminated. Typically, for example, the area of thesurface of substrate 60A that faces substrate 2 is smaller than the areaof the surface of substrate 2 by 2% or more (e.g., 3% or more, 5% ormore, 7% or more, 10% or more, 15% or more, 20% or more, 25% or more,30% or more).

Platforms 60A and 60B can be attached to blocks 80A and 80B,respectively. Block 80A includes lateral sides 81A-84A supporting a topside 85A as shown in FIG. 8. Blocks 80A and 80B can be made of the sameor similar materials to those used for the platforms, including metals,ceramics, and/or plastics. Thus, materials such as Delrin® can be usedto form blocks 80A and 80B, particularly in embodiments that implementRomanowsky staining of specimens. Other materials that can be used inembodiments include metals, and Teflon® brandpolytetrafluoroethylene-coated aluminum, steel, or titanium.

In some embodiments, platforms 60A and/or 60B can be raised as shown inFIGS. 7-9. Alternatively, in certain embodiments, platforms 60A and/or60B can be flush with the upper surface of blocks 80A and 80B,respectively. In either case, certain features of machine 1 as well assurface tension of fluids and surface energy of the platform or blockprevent excess fluids from flowing past the edges of platforms 60A/60Band/or blocks 80A/80B.

As shown in FIGS. 7 and 8, platform 60A can include offsets 70A-70D toprovide a separation between the surface of platform 60A and substrate2, and prevent substrate 2 from contacting platform 60A. Platform 60Bcan include a corresponding set of offsets 71A-71D. Offsets can includestandoffs, pins, pegs, rods, beads, walls, or other structures thatprovide separation between the surface of platform 60A and/or 60B andsubstrate 2. Offsets 70A-70D and 71A-71D ensure that the surfaces ofplatforms 60A and 60B and substrate 2 remain substantially parallel whensubstrate 2 contacts the offsets. The benefit of maintaining these twosurfaces in parallel is that the volume enclosed between these twosurfaces is thus defined and can be precisely controlled. If the twosurfaces are not substantially parallel, and the angle between themchanges, then the volume between them also changes and is not fixed andprecisely controlled. In addition, the fluids may not apply uniformly tothe specimen if such two surfaces are not substantially parallel.

As used herein, the phrase “substantially parallel” means that twosurfaces are exactly parallel or nearly parallel, so that imperfectionsin the surface flatness of substrate 2 are reduced or eliminated whensubstrate 2 contacts the offsets. For example, although great care istaken in the production of substrates, certain substrates may haveimperfections such as twist and/or non-coplanar corners. In the systemsand methods disclosed herein, the use of offsets assists in correctingthese imperfections by improving the surface flatness of substrate 2where needed, orienting substrate 2 in a substantially parallelrelationship to platforms 60A and 60B in the process. The phrase“substantially parallel” covers situations in which the two surfaces arenot perfectly flat, but the offsets are all the same size or height, sothat at least the contact points of a surface of the substrate with theoffsets are in the same plane.

FIG. 12A shows substrate 2, substrate gripper 20B, blocks 80A, 80B,platforms 60A, 60B, offsets 70A-70D and 71A-71D, and separation 92between substrate 2 and platform 60B. Separation 92 allows fluids totravel between the surface of platform 60B containing ports 40B-45B andsubstrate 2. The separation distance required for optimal specimenfixing, staining, and rinsing will vary depending on the flow rate offluids dispensed from ports 40B-45B (and/or ports 40A-45A), portdiameter, the viscosity of the fluids applied during processing, and theamount of suction available for removing fluids from the substrate,separation, and platform.

In some embodiments, for example, offsets providing a separation 92 ofabout 100-200 microns between the surface of platform 60B and substrate2 enable fixing, staining, and rinsing for specimens comprising bloodcells in embodiments capable of dispensing fluids at flow rates rangingfrom about 50 to about 300 microliters per second from ports 40B-45Bhaving a diameter ranging from 500 to 1,500 microns. In general, thesize or height of separation 92 can vary from about 50 microns to 1,000microns for certain embodiments (e.g., from about 50 to 500 microns,from about 75 to 250 microns, from about 100 to 200 microns), providedsuch embodiments are capable of overcoming surface tension from fluidsin the separation while dispensing and removing fluid during specimenprocessing. In addition, in certain embodiments, the diameters of portslocated on platform 60A and/or 60B can vary from about 125 microns to5,000 microns.

FIGS. 12B and 6C show a ball joint mechanism 25 that can be used toalign a substrate gripper 20A to be parallel with a platform 60A. Theball joint mechanism 25 can include a ball member 25A that is rigidlyfixed to the substrate gripper 20A, a deflection element 25B (e.g., aspring), a lower socket 25C that is rigidly connected to the substratearm 10A, an upper socket 25D, a cap 25E that is fixed to the lowersocket 25C (e.g., using fasteners), and a set screw 25F. In someembodiments, during manufacturing and/or set up of the machine 1 andsubstrate grippers 20A and/or 20B, the ball joint mechanism 25 can beadjusted to compensate for any misalignment that may be present due totolerance stack-up or fabrication problems. To adjust the ball jointmechanism 25, in some embodiments, the set screw 25F is loosened and thesubstrate arm 10A is moved to the closed position. Since the set screw25F is loosened, the substrate gripper 20A, while gripping a substrate2, is able to lay substantially parallel to the platform 60A while thesubstrate 2 positioned along the contact offsets 70. Alternatively, insome embodiments, the number of offsets on platform 60 can be reduced oreliminated completely; a shim with a thickness corresponding to thedesired separation distance can be used temporarily during set up orcalibration of machine 1 in conjunction with ball joint mechanism 25 toset separation 92 at a desired distance for specimen processing.Although the ball joint mechanism 25 is loosened, the deflection element25B applies a force to keep the substrate gripper 20A semi-fixed to thesubstrate arm 10A so that it is able to move independently, but it isnot so loose and not free to move so much as to interfere with, or causedamage to, other components of the machine 1. Once the substrate 2 ispressed firm in a closed position so the substrate 2 is substantiallyparallel to the platform 60A, the set screw 25F can be tightened tosecure the ball joint mechanism 25. As shown, when tightened, the setscrew 25F applies a downward force on the upper socket 25D and thusapplies a frictional force to the top of the ball member 25A via theupper socket 25D. Since the lower socket 25C is fixed to the cap 25E,the force created by the set screw 25F also lifts the lower socket 25Csuch that the lower socket 25C applies a frictional force to the bottomside of the ball member 25A to constrain the ball member 25A within theupper and lower sockets 25C, 25D. Once constrained to the ball member25A, the substrate gripper 20A becomes fixed to the substrate arm 10A.

Typically, once the substrate gripper 20A is positioned and constrainedwith the set screw 25F, the ball joint mechanism 25 need not be adjustedagain during normal use. However, if the substrate gripper 20A becomesmisaligned and therefore the ball joint mechanism 25 requires adjustment(e.g., due to damage, machine repair, poor performance, or otherreasons), the set screw 25F can be loosened, the substrate gripper 20Acan be moved to a closed position to position so that a substrategripped by the substrate gripper 20A is substantially parallel to theplatform 60A, and then set screw 25F can be tightened to secure the balljoint mechanism 25.

In general, actuators 30A and/or 30B can be configured to adjust theposition of substrate arms 10A and/or 10B to vary the extent ofseparation between the surface of platforms 60A and/or 60B and substrate2. Varying this separation provides greater flexibility in embodimentsthat allow for adjusting the fluids assigned to each port, flow rates,fluid viscosities, and evacuation forces from platforms 60A and/or 60B.For example, a 100 micron separation 92 can provide sufficient specimenfixing, staining, and rinsing when fluids applied from platform 60A aredispensed at a flow rate of 115 microliters per second from ports40A-45A having port diameters ranging from 500 microns to 1,500 microns.Alternatively, with a separation 92 distance between the surface ofplatform 60A and substrate 2 of approximately 200 microns, a higher flowrate for fluids dispensed from ports 40A-45A, such as 140 microlitersper second, can be used for specimen processing.

As disclosed above, machine 1 may contain a series of ports and tubesfor dispersing and removing fluids applied during specimen processing.The following discussion describes various ports, tubes, and othercomponents associated with platform 60A, but similar considerationsapply to platform 60B and its associated components. FIG. 8 shows aclose up view of the apparatus shown in FIG. 7, and shows in detailports 40A-45A on platform 60A and tubes 50A-55A connected to block 80A.Tubes 52A-55A distribute certain fluids including one or more fixatives,stains, and rinse solutions across the platform, into the separation,and onto the substrate. As shown in FIG. 8, the top side of platform 60Aincludes six ports 40A-45A that are connected to tubes 50A-55A. Fluidsare driven by one or more pumps through the tubes and ports ontosubstrate 2. One or more fluid reservoirs 210A-213A (such as a firststain reservoir 211A, a second stain reservoir 212A, a fixativereservoir 210A, and a rinse solution reservoir 213A), e.g., as shown inFIG. 10, can direct fluid onto platform 60A and substrate 2. Thediameters of ports 40A-45A shown in FIGS. 7-9 range from approximately500 microns to 1,500 microns, although the diameters can also be smalleror larger in certain embodiments. In some embodiments, the diameters ofthe vacuum ports 40A and 41A are more than twice the diameters of fluidports 42A-45A.

Each of ports 40A-45A is typically dedicated to a particular fluid orvacuum source. Alternatively, more than one port may be used for eachfluid or vacuum source, or multiple tubes from various fluid and vacuumsources may connect to a single port located on platform 60A. Forexample, in some embodiments, only one port on platform 60A may be usedfor waste removal, but when using more viscous fluids, the single portmay not provide sufficient suction to evacuate residual fluid from theplatform. Thus, it may be desirable in certain embodiments to providetwo suction ports at different positions on the platform (e.g., onesuction port at each end of the platform) for removing excess stain,fixative, and rinse fluids as shown with ports 40A-41A in FIG. 8.Further highlighting the variability of fluid-to-port configurations, incertain embodiments, a single port on platform 60A may be dedicated fora particular stain, while in other embodiments multiple ports are usedfor applying stains during specimen processing. Indeed, variouscombinations relating to the number of ports, port locations, and fluidsassigned to each port and fluid tube may be used in differentembodiments of the invention.

Ports 40A-45A can generally be positioned as desired on platform 60A toprovide for fluid delivery to, and fluid removal from, substrate 2.Typically, each of the fluid ports is positioned on platform 60A suchthat the port's aperture is not positioned directly adjacent or beneathspecimen 3 on substrate 2 when the specimen is undergoing processing.With certain combinations of specimens and stains, for example, ifstains are dispensed from a port located directly adjacent or beneath aportion of specimen 3, a larger quantity of stain may be applied tocells in that portion (in the vicinity of the port) than to cells inother portions of the specimen. As a result, cells receiving the largerquantity of stain may appear darker in specimen images, and thisnon-uniform staining of specimen cells can introduce errors intodiagnostic measurements and analytical outcomes based on the images.Thus, fluid ports that deliver stain to specimen 3 can be spaced acertain distance from the specimen-containing area of a substrate (e.g.,a slide) to improve staining results.

In addition, the use of pairs of ports, e.g., multiple pairs of ports,located opposite each other, can also improve staining uniformity. Forexample, in some embodiments, two ports are used to deliver stain tospecimen 3. The two ports can be located on platform 60A at positionsspaced a certain distance (e.g., are offset) from the edges of specimen3, and located opposite each other in a direction parallel to the shortedges of platform 60A. When stain is dispensed from the two spacedports, a relatively uniform quantity of stain is deposited on the cellsin different regions of specimen 3, and improved staining homogeneity isobserved in specimen images.

Similarly, while ports 40A-45A can generally be positioned as desired toremove excess fluids from the surface of substrate 2 using one or morevacuum sources, in some embodiments ports that are used for fluidremoval are spaced at a distance from positions on platform 60A that aredirectly beneath cells within specimen 3 on substrate 2. Positioningwaste removal ports in this manner reduces the chances that when suchports are actuated to evacuate fluids from substrate 2, cells fromspecimen 3 are inadvertently also drawn into the fluid removal ports. Incertain embodiments, due to the difference in lengths of the long andshort sides of platform 60A, the waste removal ports are spaced apartfrom the edge of the specimen area and arranged opposite each otheralong a direction parallel to the long edges of platform 60A.

Machine 1 can include or connect to a control system 5 as shown in FIG.10, which provides another perspective view of machine 1. Control system5 can include one or more computers each containing a central processingunit capable of executing software instructions stored on computerreadable media such as a hard drive, optical drive, or memory.Additionally, control system 5 can include electrical circuitry forexecuting the software instructions. Control system 5 can include a userinterface for receiving user commands to control the operation ofmachine 1. Software stored on or provided to the computer can includeprograms that control the operation of components of machine 1, such asfluid pumps and vacuums, during specimen processing. For example, thesoftware can include instructions for directing the machine 1 to applyvarious fixatives, stains, and rinses to the specimen, and to performseveral agitation steps during specimen processing.

In addition, the software can include default settings, and the userinterface may contain customization features for providing the user withthe ability to change these defaults settings. For example, the userinterface can contain customization features for allowing a user tocustomize the speed, frequency, or order of fixing, staining, andrinsing phases, as well as agitation parameters (further describedbelow). Control system 5 can also communicate via a network protocol(such as Appletalk®, IPX, or TCP/IP). For example, the network protocolmay use cables (such as twisted pair cables) and/or a wirelessconnection such as WiFi. The control system may be connected to alaboratory information system using the network protocol. The laboratoryinformation system can contain a server and/or database for storinginformation relating to specimens processed on machine 1. For example,the database may contain a table that provides information about theperson or source of the specimen (e.g., name, date of birth (DOB),address, time specimen was taken, gender, etc.), information relating toprocessing of specimen (processed on date ##/##/####, specimen number #,etc.), a copy of any images acquired of the specimen, and copies of anyresults obtained by analyzing the images.

FIG. 7 also shows that machine 1 can include supports 110A and 110B tosecure the device to a location within a system or a laboratoryworkstation. Machine 1 also includes one or more substrate arms 10A and10B, each connected at their base to an actuator 30A and 30B. Theopposite ends of the substrate arms 10A and 10B include substrategrippers 20A and 20B for receiving and holding substrates duringspecimen processing. Each substrate gripper 20A and 20B receives andholds a substrate 2 while machine 1 completes all specimen processingsteps (described below). The substrate may be or include a microscopeslide, a cover slip, or other transparent material suitable for holdinga specimen during specimen processing and microscopic examination afterspecimen processing. The embodiment of FIG. 7 depicts a glass microscopeslide, substrate 2, which includes a specimen 3. Using suction ports,substrate grippers 20A, 20B can hold the substrate 2 to substrate arms10A, 10B during specimen processing. A suction tube 23 provides suctionto the substrate grippers 20A and 20B through suction ports 21A and 21B,and 22A and 22B (note that ports 21A and 22A are positioned behind theslide 2 in FIG. 7, and are shown in dashed lines).

The machine 1 embodiment shown in FIGS. 7-9 is a dual substrate machine,capable of holding and processing a substrate on each of substrate arms10A and 10B. Other embodiments provide for processing a single substrateor three or more substrates, sequentially or simultaneously. Further,while the embodiments depicted in FIGS. 7-12 use suction to attach thesubstrates 2 to the substrate arms 10A and 10B, alternative embodimentsuse various types of clamps, fingers, or magnets (if the substrate ismagnetized) to attach a substrate 2 to a substrate arm 10A duringspecimen processing.

In the embodiments shown in FIGS. 11 and 24A-B, machine 1 receives asubstrate 2 carrying a specimen 3 from an automated substrate mover 120or manually from an individual. As an example, the substrate mover 120can be a device that transports a substrate between stations (e.g.,station 121 to station 122 to station 123, to station 124, and tostation 125). FIG. 11 shows a system having a first label reader station121, an applicator station 122, a staining station 123 that includesmachine 1, a camera or imaging station 124, and a second label readerstation 125. The first label reader station 121 is configured to readinformation from substrate 2 such as a bar code and/or “fingerprint”information that is used to identify the particular substrate 2 andspecimen 3 thereon. The second label reader station 125 functions in thesame manner, and the information it reads is used to verify that thespecimen 3 that is imaged at station 124 is the same as the substratethat was processed.

Substrate mover 120 can include a gripper 127 for holding the substrate2, and registration circuitry or software to enable the mover 120 todetermine whether the substrate 2 is mounted in the mover 120. In oneembodiment, substrate mover 120 can include a hydraulic cylinder formoving substrate 2 from a first station 121 to a second station 122.After specimen processing, the substrate mover 120 may remove theprocessed substrate from staining station 123 and transport thesubstrate 2 to another station for substrate examination, such as amicroscope or station 124. Alternatively, an individual may manuallyremove a substrate from machine 1 after specimen processing.

The substrate arms 10A and 10B can rotate about an axis to enable thesubstrate to move from an open position for loading, to a specimenprocessing position, and back to the open position for unloading afterspecimen processing. FIG. 13A shows a flow chart 500 that includes aseries of steps for moving substrate arms from an open position to aprocessing position. Flow chart 500 is further described below withreference to FIG. 13B, which shows a schematic diagram of machine 1.

Note that machine 1 in FIG. 7 is configured to accept and examine twosubstrates. In the following discussion and figures, reference may bemade to only one set of components in machine 1 (e.g., substrate gripper20A, actuator 30A, substrate arm 10A, etc.). However, it is to beunderstood that the same steps, features, and attributes that aredisclosed in connection with one set of components can also apply to theother set of components in machine 1 (e.g., substrate gripper 20B,actuator 30B, substrate arm 10B, etc.). Thus, while the discussionherein focuses only on one set of components for clarity and brevity, itis understood that machines for specimen examination such as machine 1can include two or even more than two sets of components, each sethaving some or all of the features discussed herein.

Returning to FIGS. 13A and 13B, in a first step 502 of flow chart 500,substrate mover 120 places a substrate 2 in contact with a substrategripper 20A. In step 504, substrate 2 is positioned on the substrategripper in a “specimen up” or “open” position. Next, in step 506,actuator 30A rotates substrate arm 10A by approximately 180° (see FIG.13B) to position substrate 2 in a “specimen down” or “specimenprocessing” or “closed” position (step 508), directly above platform60A, so that substrate 2 is in a processing position in step 510.

Then, in step 512, machine 1 stains specimen 3 positioned on substrate 2by directing suitable fluids including stains, rinse fluids, andfixatives to be pumped from reservoirs 210A, 211A, 212A, and 213A intocontact with specimen 3 through ports 42A, 43A, 44A, and 45A. Excessfluids are removed from specimen 3 by vacuum pumping through ports 40Aand 41A, and are collected in waste collectors 230 and 231.

In step 514, following staining of specimen 3, actuator 30A rotatessubstrate arm 10 by approximately 180° (reversing the rotation of step506) to return the substrate to the “specimen up” position. Finally, instep 516, substrate mover 120 removes the processed substrate fromsubstrate gripper 20A. Other open or “specimen up” positions can also beused, provided that an operator or automated substrate mover can loadand unload substrates from machine 1. For example, the specimen upposition can be rotated 100° or more (e.g., 120° or more, 130° or more,140° or more) from the specimen processing position. In someembodiments, the specimen up position can be rotated less than 100°(e.g., less than 90°, less than 80°, less than 70°) from the specimenprocessing position, provided that an operator or substrate mover canload and unload substrates from machine 1.

Actuators 30A and/or 30B may include an electric motor, pneumatics,magnetic systems, or other hardware (e.g., a worm gear) to move arm 10Aand/or 10B. When substrate arms 10A and 10B are in an open position asdepicted in FIG. 7, grippers 20A and 20B can each receive a substrate 2.Once loaded onto a substrate gripper 20A or 20B, actuators 30A and/or30B then rotate arms 10A and/or 10B, and thus substrate 2, from the open(“specimen up”) position to a processing position (“specimen down,” asshown for arm 10B in FIG. 9) for application of fixative, stain, andrinse including agitation steps, and back to an open position forunloading after processing.

With reference to FIG. 9A, actuator 30B has rotated substrate arm 10Bfrom the open position depicted in FIG. 1 to a “closed” or processingposition. FIG. 9A shows that the substrate 2 on substrate arm 10B hasbeen flipped over and rotated approximately 180° from its loadingposition shown in FIG. 7 to a downward-facing position where specimen 3on substrate 2 is substantially parallel to the surface of platform 60B.As discussed in connection with FIG. 13A above, while substrate 2 ispositioned proximal to platform 60B in the specimen processing positionshown, machine 1 applies various fixatives, stains, and rinses tospecimen 3 on substrate 2 through several processing phases, which willbe described in greater detail below. To remove substrate 2 from theprocessing position, actuator 30B rotates substrate arm 10B back to theopen position shown in FIG. 7 (both arms) and FIG. 9A (where only arm10A is in the open position).

In certain embodiments, control system 5 can detect the position of thearms utilizing one or more sensors 105A and 105B to detect indicatorarms 101A and 101B (as shown in FIGS. 7 and 9). Sensors 105A and 105Bcan be proximity sensors, e.g., photoelectric sensors, utilizing, e.g.,infrared light or various other technologies (lasers, motion detectors,etc.) to detect the presence or absence of the arms. For example,proximity sensors 105A or 105B can have a detection field, and thesensors can determine whether or not a substrate arm (e.g., arm 10Aand/or 10B) or a substrate gripper (e.g., gripper 20A and/or 20B) iswithin the detection field by detecting indicator arm 101A and/or 101B.Control system 5 can receive information from the sensors to determinethe positions of substrate arms 10. For example, when substrate arm 10B(not shown in FIG. 9) is rotated to a processing position, proximitysensor 105B on the proximal end of indicator arm 101B senses targetsubstrate gripper 20B, and notifies control system 5 that substrate arm10B is rotated to a specimen processing position. In this position,proximity sensor 105B on the distal end of indicator arm 101B will notsend a signal to control system 5, because the sensor does not detectany target (e.g., a substrate arm or substrate gripper).

When substrate arm 10B rotates to an open position (as shown in FIG. 7),proximity sensor 105B on the distal end of indicator arm 101B sensestarget substrate gripper 20B, and notifies control system 5 thatsubstrate arm 10B is rotated to an open position. Stated differently,when substrate arm 10B has rotated away from the sensor 105B, thesensors send a “not present” signal to the control system 5. When arm10B is rotated into the open position, arm 10B is closer to the sensor105B, and the sensor can send a “present” signal to the control system5. In alternate configurations, the sensor can be mounted on substrate10B and can detect the presence of the indicator arm 101B. In someembodiments, control system 5 can be used to calibrate the position ofactuators 30A and 30B to known open and specimen processing positions,and/or to actively monitor the movement and position of substrate arms10A and 10B based on control signals and/or feedback received fromactuators 30A and 30B.

The structure and axis of rotation for substrate arms 10A and 10B inFIG. 7 may be varied in other embodiments of the invention. FIG. 14Ashows a flow chart 600 that includes an alternate series of steps formoving substrate arms from an open position to a processing position.Flow chart 600 is further described below with reference to FIG. 14B,which shows a schematic diagram of machine 1.

In step 602 of flow chart 600, substrate mover 120 places substrate 2 onsubstrate gripper 20A in a “specimen up” orientation. Then, in step 604,a first actuator 30A rotates substrate 2 by approximately 180° in aplane perpendicular to the plane of FIG. 14B, so that substrate 2remains oriented in a “specimen up” position above platform 60A. In step606, a second actuator 35A receives substrate 2 oriented in the“specimen up” position. Then, in step 608, second actuator 35A (e.g.,positioned between substrate arm 10A and substrate gripper 20A) rotatesthe substrate 2 into a “specimen down” orientation. Second actuator 35Acan also move substrate 2 downward toward platform 60A so that substrate2 contacts offsets 70A and 70B.

Next, with substrate 2 in the processing position in step 610, machine 1stains specimen 3 on substrate 2 by applying stains, fixatives, andrinse solutions as discussed above in connection with step 512 of flowchart 500. After staining is complete, second actuator 35A rotatessubstrate 2 from a “specimen down” orientation to a “specimen up”orientation (step 614), and then first actuator 30A rotates substrate 2by approximately 180° (e.g., in a plane perpendicular to the plane ofFIG. 14B, reversing the rotation applied in step 606) so that thesubstrate remains oriented in a “specimen up” position. Finally, in step618, substrate mover 120 removes the processed substrate from substrategripper 20A.

Fixative Phases

Referring to FIG. 10, fluid tubes 52A-55A and 52B-55B can be positionedto deliver fixative to platforms 60A and 60B, separation 92, substrate2, and specimen 3 during specimen processing. One or more fluid tubes52-55A can be connected to a port inside platform 60A and a respectivefixative reservoir 210A. The fluid tubes may also include a connectionto a pump 200A and/or a valve capable of directing fixatives from thereservoir through the tube and a port located on the platform, and ontoa substrate and specimen. As an example, pump 200A can direct a fixativesolution through tube 54A, out of block 80A, through port 44A, ontoplatform 60A, into the separation 92 between the platform 60A andsubstrate 2, and onto substrate 2 containing specimen 3. After applyinga specific quantity of the fixative solution to substrate 2, a vacuum orother suction source 220A and/or 221A can evacuate residual fixativesolution from platform 60A, the separation 92, and substrate 2 intowaste container 230A and/or 231A via one or more of ports 40A and/or 41Athrough waste tubes 50A and 51A.

FIG. 15 shows a flow chart 700 that includes a series of steps forapplying a fixative solution to a specimen. In step 702, a pump (e.g.,pump 200A) directs the fixative solution (e.g., methanol) from areservoir (e.g., reservoir 210A) into a fixative solution tube (e.g.,tube 54A). In step 704, the fixative solution is directed into port 44Aattached to block 80A. Then, in step 706, the fixative solution isdirected out of port 44A in platform 60A. In step 708, the fixativesolution is directed out through port 44A and into separation 92 betweensubstrate 2 and platform 60A. Finally, in step 710, specimen 3 onsubstrate 2 is fixed by the fixative solution.

In some embodiments, pump 200A directs the fixative solution throughtube 54A and port 44A, onto platform 60A and into the separation 92 at aflow rate of about 50 μL or more (e.g., 75 μL or more, 100 μL or more,150 μL or more, 200 μL or more, or 250 μL or more) and/or about 300 μLor less (e.g., 250 μL or less, 200 μL or less, 150 μL or less, 100 μL orless, or 75 μL or less) microliters per second for a period of about two(e.g., three, four, or five) seconds. For example, the flow rate can beabout 115 μL/s (e.g., about 70 μL/s, about 100 μL/s, or about 150 μL/s)for a period of about two seconds (e.g., three seconds, or fourseconds). A vacuum or other suction source 220A and/or 221A then removesresidual fixative solution present in separation 92 and/or on theplatform 60A and substrate 2 using ports 40A and/or 41A and waste tubes50A and/or 51A (further described below). Next, the pump 200A can againdirect the fixative solution through tube 54A and port 44A, and ontoplatform 60A at a flow rate of about 50 μL or more (e.g., 75 μL or more,100 μL or more, 150 μL or more, 200 μL or more, or 250 μL or more)and/or about 300 μL or less (e.g., 250 μL or less, 200 μL or less, 150μL or less, 100 μL or less, or 75 μL or less) microliters per second fora period of about two (e.g., three, four, or five) seconds. For example,the flow rate can be about 115 μL/s (e.g., about 70 μL/s, about 100μL/s, or about 150 μL/s) for a period of about two seconds (e.g., threeseconds, or four seconds). This process of fixing and evacuating can berepeated again, using the same or a different fixative, depending on thetype of specimen requiring fixation. Further, machine 1 is capable ofvarying the frequency and flow rates for each fixing phase. Other flowrates sufficient to overcome any surface tension in the fluid located inseparation 92 can also be used. By adjusting the frequency and/or flowrate of the fixing phases, machine 1 can achieve optimal fixation forvarious specimens using several different fixatives. Machineinstructions for different types of specimens can be hardwired orpreprogrammed in control unit 5 and selected by a system operator asneeded.

In general, a wide variety of fixatives can be applied to specimensduring fixative phases. For example, 85% methanol can be used as thefixative. For some stains, an ethyl alcohol or formaldehyde basedfixative can be used. Embodiments of the fixative solutions disclosedherein can be used to prepare a specimen for examination.

Staining Phases

Machine 1 also includes tubes and ports configured to apply one or moredyes or stains to a specimen fixed to a substrate in one or morestaining phases. Staining a specimen increases the contrast of thespecimen when it is viewed or imaged under a microscope or other imagingdevice.

FIG. 16 is a flow chart 800 that includes a series of steps for applyingstain to a specimen. In step 802, a pump (e.g., pump 201A) directs astain solution from a reservoir (e.g., reservoir 211A) into a stain tube(e.g., tube 52A). In step 804, the stain solution is directed into aport (e.g., port 42A) attached to block 80A. Next, in step 806, thestain solution flows out of port 42A in platform 60A. In step 808, thestain solution flows into separation 92 between substrate 2 and platform60A. Finally, in step 810, the stain is applied to specimen 3 onsubstrate 2.

In some embodiments, multiple tubes and ports can be used to apply stainto specimen 3. For example, a second pump (e.g., pump 202A) can direct astain solution (e.g., the same stain or a different stain from thatdispensed from reservoir 211A) from reservoir 212A through tube 53A andport 43A and onto platform 60A. In certain embodiments, two or morefluid tubes may connect to a shared stain reservoir or pump and/or valveused to direct stain through the ports and onto the platform. Referringback to FIG. 8, tube 52A may deliver red stain, such as eosin Y, to theplatform, substrate 3, and specimen 2. Tube 53A may deliver blue stain,such as a thiazine dye (e.g., azure B, methylene blue). In FIGS. 7-12,the numbers, locations, and sizes of the ports on platform 60A areselected to optimize the application of stain to a specimen fixed to thesubstrate. If other stains are selected, a different number, locations,and sizes of ports may be preferable depending on the viscosity of thestain.

Each of ports 40A-45A (and 40B-45B) can include both an input channelfor receiving fluid and an output channel for outputting fluid. In someembodiments, the output channels of the rinse 45A, fixative 44A, andstaining ports 42A-43A are on the upper surface of platform 60A, and theinput channels of vacuum ports 40A and 41A may be on opposite ends ofthe upper surface of platform 60A. The input channels of the rinse 45A,fixative 44A, and staining ports 42A-43A may be situated on the samelateral side of block 80A, and the output channels of the vacuum ports40A and 41A can be positioned on opposite lateral sides of block 80A.

By way of example and with reference to FIGS. 8 and 16, control system 5instructs a pump (e.g., pump 201A) in step 802 to direct a first stainsolution (e.g., a stain comprising eosin Y) from a stain reservoir intofluid tube 52A. In step 804, the first stain solution enters port 42Afrom the fluid tube. Then, in step 806, the first stain solution leavesport 42A, and in step 808, the first stain solution is deposited intoseparation 92 between platform 60A and substrate 2 at a flow rate ofabout 50 μL or more (e.g., 75 μL or more, 100 μL or more, 150 μL ormore, 200 μL or more, or 250 μL or more) and/or about 300 μL or less(e.g., 250 μL or less, 200 μL or less, 150 μL or less, 100 μL or less,or 75 μL or less) microliters per second for a period of about two(e.g., three, four, or five) seconds. For example, the flow rate can beabout 115 μL/s (e.g., about 70 μL/s, about 100 μL/s, or about 150 μL/s)for a period of about two seconds (e.g., three seconds, or fourseconds). In step 810, specimen 3 on substrate 2 is stained with thefirst stain solution. Following staining, a vacuum or other suctionsource (e.g., pumps 220 and/or 221) may then evacuate residual firststain solution present in separation 92, on platform 60A, and onsubstrate 3 using ports 40A-41A and waste tubes 50A-51A.

Machine 1 can be programmed to repeat these staining and evacuationphases after a delay (e.g., a delay of between 3 seconds and 10 seconds,such as a five second delay), following the first staining phase. Asecond pump 202A can be instructed by control system 5 to direct secondstain solution (a solution comprising a thiazine dye) from a stainreservoir through fluid tube 53A, out port 43A at a flow rate of about50 μL or more (e.g., 75 μL or more, 100 μL or more, 150 μL or more, 200μL or more, or 250 μL or more) and/or about 300 μL or less (e.g., 250 μLor less, 200 μL or less, 150 μL or less, 100 μL or less, or 75 μL orless) microliters per second for a period of about two (e.g., three,four, or five) seconds onto platform 60A. For example, the flow rate canbe about 115 μL/s (e.g., about 70 μL/s, about 100 μL/s, or about 150μL/s) for a period of about two seconds (e.g., three seconds, or fourseconds). A vacuum or other suction source (e.g., pump 220A and/or 221)may then evacuate residual second stain solution present in separation92 and/or on platform 60A and/or on substrate 2 using ports 40A-41A andwaste tubes 50A-51A. As with the fixing phases, machine 1 is capable ofvarying the frequency and flow rates for each staining phase. The flowrate may range, e.g., from 50 to 300 microliters per second, or may besmaller or greater than the outer limits of this range (e.g., 10 to 500microliters per second) provided the flow rate is sufficient to overcomeany surface tension present in the fluid located in separation 92.

Exemplary stains that can be applied to specimens include, but are notlimited to: Wright-Giemsa stain, Giemsa stains, and Romanowsky stains,including embodiments of the first and second staining solutionsdisclosed herein. Other agents such immunocytochemical reagents or othermarkers of specific cell components can also be applied to specimens.

Waste Fluid Removal

As referenced above, a vacuum or other suction source 220 and/or 221 canevacuate residual fluid from substrate 2, separation 92, and platform60A during or between fixing and staining phases. Referring to FIG. 7,one or more waste tubes can be connected to sides 82A and 84A of block80A. Waste or vacuum tubes 50A and 51A are used to withdraw fluid andsmall particulate matter from platform 60A, separation 92, and substrate2 into a waste container or other location separate from machine 1. Withreference to FIG. 8, waste tubes 51A and 51B may be connected toseparate vacuum sources 220 and 221, and waste containers 230 and 231,at the distal ends of the waste tubes. Alternatively, two or more wastetubes can be connected to a single vacuum source, and the same wastecontainer, as shown in FIG. 10. Waste tubes 50A and 50B may extendthrough pinch valves 90A and 90B, respectively.

A vacuum or other source (e.g., vacuum pump 220 and/or 221) for applyingsuction may be connected to one or more of waste tubes 50A, 50B, 51A,and 51B to draw fluid from the platforms 60A and/or 60B, separation 92,and substrate 2 into waste containers 230 and 231. The vacuum forceapplied within the waste tubes may be equivalent to negative one tonegative five pounds per square inch (“psi”) to provide sufficientsuction for removing fluids when the separation between the substrate 2and the platform is between 100 to 200 microns. In general, as usedherein, “negative” pressure refers to a pressure less than the ambientpressure within machine 1 or the environment surrounding machine 1. Forexample, in some embodiments, the environment surrounding machine 1 hasan ambient air pressure of approximately one atmosphere. “Negative”pressures refer to pressures that are less than this ambient airpressure (e.g., a pressure of negative one psi applied to a fluid is apressure of one psi less than the ambient air pressure exerted on thefluid). Other vacuums ranging from 0.1 psi to 14 psi, or greater, can beused provided such vacuums are sufficient to overcome any surfacetension in the fluid present in the separation. In addition, immediatelyprior to applying vacuum to evacuate fluids from the separation,actuator 30A can raise the proximate edge of substrate 2 a distance of15-35 microns from the specimen processing position. This increasedseparation between substrate 2 and platform 60 can improve evacuation ofany residual fluids in separation 92 during a vacuum phase.

In some embodiments, control system 5 is configured to vary thefrequency and vacuum applied for fluid removal during specimenprocessing. FIG. 17A includes a flow chart 900 that features a series ofsteps for removing excess fluid from a substrate. Following a fixingphase, for example, control system 5 can open pinch valves 90A and/or90C in step 902 and apply a vacuum of 5 psi in the waste tubes (e.g.,waste tubes 50A and 51A) for a five second period. During this period, afixative solution is removed (step 904) from the separation, substrate,and platform through ports 40A and 41A. The fluid travels through thewaste tubes in step 906, and is deposited in into one or more wastecontainers (e.g., containers 230 and/or 231) in step 908. Once theevacuation period expires, control system 5 can instruct one or more ofthe pinch valves 90A, 90C to close off the waste tubes 50A and/or 51A instep 910, thereby preventing further evacuation by the vacuum 220-221.Control system 5 may direct machine 1 to repeat this fluid removal stepafter each fixing phase.

FIG. 17B includes a flow chart 1000 that features an alternate series ofsteps for removing excess fluid from a substrate. The method in flowchart 1000 does not use pinch valves to seal waste tubes. Instead, aftera staining phase, suction source 220 and/or 221 are initialized in step1002 and enter an active state in step 1004. The suction source appliesa pressure of 3 psi in waste tubes 50A and/or 51A for a four secondperiod to remove stain from separation 92, substrate 2, and platform 60Athrough ports 40A and 41A in step 1006. The evacuated fluid travelsthrough waste tubes 50A and/or 51A in step 1008, and is deposited in oneor more waste containers 230, 231 in step 1010. Machine 1 may repeatthis fluid removal step after each staining phase. By varying thefrequency and pressure applied during fluid removal steps, machine 1 mayachieve optimal fixing and staining of specimens.

Pinch valves 90A, 90B, 90C, and 90D close off waste tubes 50A, 50B, 51A,and 51B, as shown in FIG. 7. The pinch valves 90A-90D may bemechanically, electrically, hydraulically, or pneumatically actuatedthrough actuators contained within or external to the valves. Pinchvalves 90A-90D operate to prohibit fluid flow through waste tubes 50A,50B, 51A, and 51B. For example, when changing or emptying a full wastecontainer 230 from machine 1, it may be desirable to close the pinchvalves (90A-90D) to prevent leakage of residual fluids present in thewaste tubes. Different valve types or other mechanisms such as clamps orstoppers may be used with embodiments of machine 1 to close the wastetubes 50A, 50B, 51A, and 51B.

Rinsing Phases

Rinse solutions can be applied during specimen processing with machine 1in one or more rinse phases. For example, it may be desirable to cleansethe substrate 2, separation 92, and platforms 60A and/or 60B betweenfixing phases, between staining phases, and/or between fixing andstaining phases.

FIG. 18 includes a flow chart 1100 featuring a series of steps forrinsing a specimen. In step 1102, a pump (e.g., pump 203A) directs rinsesolution (e.g., comprising distilled water) from a reservoir (e.g.,reservoir 213A) into a rinse tube (e.g., rinse tube 55A). In step 1104,the rinse solution enters port 45A connected to block 80A. In step 1106,the rinse solution flows onto platform 60A through the output channel ofport 45A, and in step 1108, the rinse solution enters separation 92between substrate 2 and platform 60A. In step 1110, rinsing of specimen3 is performed. Finally, in step 1112, a vacuum source 220, 221 appliessuction to one or more of waste tubes 50A and 51A to remove rinsesolution from separation 92 and substrate 2; the rinse solution istransported to waste container 230 and/or 231. In some embodiments, theforegoing steps are repeated in a second rinse phase.

In some embodiments, control system 5 may direct pump 203A to apply therinse solution at a flow rate of about 50 μL or more (e.g., 75 μL ormore, 100 μL or more, 150 μL or more, 200 μL or more, or 250 μL or more)and/or about 300 μL or less (e.g., 250 μL or less, 200 μL or less, 150μL or less, 100 μL or less, or 75 μL or less) microliters per second fora period of about two (e.g., three, four, or five) seconds. For example,the flow rate can be about 115 μL/s (e.g., about 70 μL/s, about 100μL/s, or about 150 μL/s) for a period of about two seconds (e.g., threeseconds, or four seconds). As with fixing phases, control system 5 mayvary the duration and flow rate of each rinse phase and the number ofrinse phases. In addition, control system 5 may adjust the placement ofone or more rinse phases during specimen processing. Control system 5may, for example, direct that a rinse phase occur once, after completionof all fixing phases, and that a second rinse phase occur once, aftercompletion of all staining phases. Alternatively, rinse phases may beinterspersed between two or more fixing phases or between two or morestaining phases.

In some embodiments, a staining procedure can include (1) a fixingphase, (2) a second fixing phase, (3) a first staining phase using thefirst staining solution, (4) a second staining phase using the secondstaining solution, (5) a rinsing phase, and (6) a second rinsing phase.In some embodiments, the various (e.g., first and second) stainingphases and/or solutions can be used in any order and/or repeated in anyorder. For example, the first and second staining phases and/orsolutions can be interchanged. Each phase can include a deposition flowrate of about 50 μL or more (e.g., 75 μL or more, 100 μL or more, 150 μLor more, 200 μL or more, or 250 μL or more) and/or about 300 μL or less(e.g., 250 μL or less, 200 μL or less, 150 μL or less, 100 μL or less,or 75 μL or less) microliters per second for a period of about two(e.g., three, four, or five) seconds. For example, the flow rate can beabout 115 μL/s (e.g., about 70 μL/s, about 100 μL/s, or about 150 μL/s)for a period of about two seconds (e.g., three seconds, or fourseconds).

Agitation Phases

Specimen processing in certain embodiments may include one or moreagitation phases to disperse a fixative solution, stain solution, and/orrinse solutions throughout separation 92, substrate 2, and platforms 60Aand/or 60B during the fixing, staining, and/or rinsing phases. FIG. 19includes flow chart 1200 that features a series of steps for agitating aspecimen. Actuator 30A and/or 30B, shown in FIG. 9, can provide finemovement adjustment for changing the position of substrate 2 relative toplatform 60A and/or 60B.

Control system 5 can include software and/or hardware for instructingthe actuator 30A and/or 30B to initiate an agitation phase. Actuator 30Aand/or 30B can be configured to move substrate arm 20A and/or 20B up anddown upon an agitation initiation command from the control system. Theagitation phase may repeat for a predetermined number of cycles. Theterm “cycle,” as used herein, refers to motion from a starting positionin an upward direction, followed by movement in a downward directionopposite to the upward direction. In some embodiments, one or moreagitation cycles return substrate 2 to the starting position at theconclusion of each cycle, or at least at the conclusion of some cycles.In certain embodiments, substrate 2 does not return to the startingposition at the conclusion of some or all of the agitation cycles, buteach cycle still includes an upward motion followed by a downwardmotion. Actuator 30A and/or 30B typically continues moving substrate 2in one or more agitation cycles until a stop command is sent to theactuator from the control system 5. An agitation phase may temporarilyincrease the separation size (separation distance) between substrate 2and the surface of platform 60A and/or 60B, and then return thesubstrate to the specimen processing position. In addition, an agitationphase may include a series of movements that shift substrate 2 betweenan angular position relative to the surface of platform 60A and/or 60Band the specimen processing position. Surface tension in the fluidsdispensed into the separation between the platform and substrate 2causes a redistribution of fluid molecules on the substrate when thesubstrate moves from the specimen processing position during theagitation phase and can advantageously improve fluid distribution acrossthe specimen.

Other methods can also be used to move substrate 2 relative to theplatforms during agitation phases. For example, in some embodiments, thepositions of one or more of offsets 70A-D and/or 71A-D (e.g., the amountby which the offsets extend above the surfaces of platforms 60A and/or60B) can be rapidly adjusted to agitate specimen 3. In certainembodiments, the positions of platforms 60A and/or 60B can be adjustedto cause agitation of specimen 3. For example, platforms 60A and/or 60Bcan be moved alternately up and down (e.g., corresponding to thedirection of movement of substrate 2 described above) to cause agitationof specimen 3.

In some embodiments, agitation of specimen 3 can be effected by varyingthe extent to which actuator 30A and/or 30B drives substrate 2 towardsoffsets 70A-D and/or 71A-D when the substrate arms are made of amaterial that flexes, as discussed below. Strain gauges can be used tomeasure and adjust the frequency of the agitation applied to substrate 2by detecting the variation in strain in the substrate arms as a functionof time.

Referring to FIG. 19, in a first step 1202, an agitation phase isinitiated. In step 1204, control system 5 instructs actuator 30A tobegin an agitation cycle. In response to this instruction, actuator 30Arotates substrate 2 upward in step 1206, increasing the distance betweensubstrate 2 and platform 60A. Then, in step 1208, actuator 30A rotatessubstrate 2 downward toward platform 60A, reducing the distance betweenthe substrate and platform 60A. In decision step 1210, if the agitationphase is to continue, control returns to step 1204 and the rotation ofsubstrate 2 by actuator 30A occurs again in another agitation cycle. Ifthe agitation phase is to terminate, then control passes from step 1210to step 1212, where substrate 2 is returned to its initial position withagitation complete.

The agitation phase can include one or more agitation cycles appliedthrough actuator 30A and/or 30B. Further, agitation phases can occuronce or multiple times during each of the fixative solution, stainsolutions, and/or rinse phases and in varying frequencies between eachof the fixing, staining, and/or rinsing phases. For example, andreferring to FIG. 9, actuator 30A and/or 30B may raise the proximateedge of substrate 2 vertically a distance of 35 microns from thespecimen processing position and subsequently return substrate 2 to thespecimen processing position three times, once after each fixing,staining, and rinse phase. Actuator 30A and/or 30B may complete eachagitation cycle in two seconds (e.g., one second to raise the proximateedge of substrate 2 vertically a distance of 35 microns from thespecimen processing position and one second to return the substrate tothe specimen processing position). Machine 1 is capable of carrying outinstructions to vary the agitation frequency and distance for eachagitation cycle and/or phase. For example, an agitation phase mayinclude actuator 30A and/or 30B raising the proximate edge of substrate2 vertically a distance of 5 microns from the specimen processingposition and then returning the substrate to the specimen processingposition, 10 to 20 times per second.

Alternative combinations of agitation distances and frequencies can alsobe used. For example, in some embodiments, the agitation distance is 25microns or more (e.g., 50 microns or more, 100 microns or more, 150microns or more, 200 microns or more, 250 microns or more, 300 micronsor more, 500 microns or more, 700 microns or more, 1 mm or more. Forexample, in certain embodiments, the agitation distance is between 35microns and 350 microns.

In some embodiments, the agitation cycle frequency is one cycle persecond or more (e.g., two cycles per second or more, three cycles persecond or more, four cycles per second or more, five cycles per secondor more, seven cycles per second or more, ten cycles per second ormore).

Additional agitation techniques can also be used. For example, in someembodiments, substrate gripper 20A and/or 20B may include an actuatorthat rotates the substrate about an axis perpendicular to the rotationalaxis of actuator 30A and/or 30B depicted in FIGS. 7 and 9.

Alternatively, platform 60A and/or 60B may be equipped with an offsetadjuster for raising or lowering the one or more offsets 70A-D and/or71A-D during fixing, staining, and rinsing phases. To implement theoffset adjuster, platform 60A and/or 60B can include offsets that areattached to an internal plate in the platform. The height of the platemay be varied using an internal actuator, thus varying the height of theoffsets. Alternatively, the position of the offsets 70A-D and 71A-Drelative to substrate 2 can be changed by instructing the actuator tomove platform 60A and/or 60B, or block 80A and/or 80B, thereby changingthe separation distance during the agitation phase. Control system 5 canadjust the frequency of fluid cycles, flow rate, offset height,separation distance, and agitation parameters and frequency to processspecimens more efficiently, using significantly less fluid volumesduring the specimen preparation process as compared to conventionalstaining and preparing techniques.

In some embodiments, substrate arms may be made of a material thatflexes such that if a substrate in the specimen processing positionrests against only two offsets extending from the platform, an actuatoror other motive force element may rotate the slide further towards theplatform surface until the slide rests against all four offsets. Varyingthe position of the substrate between these two positions may accomplishsufficient agitation during specimen processing. Substrate arms mayinclude strain gauges to monitor the strain in the substrate arm, andmay be used to inform control system 5 of the position of the substraterelative to the platform offsets. In addition, the control system mayinclude information corresponding to the thickness imperfections of thesubstrate, which the control systems may account for when placing thesubstrate in the specimen processing position or during agitationphases.

Drying Phases

In certain embodiments, the control system 5 can dry the specimen usinga dryer 4 attached to machine 1. FIG. 20 includes a flow chart 1300 thatfeatures a series of steps for drying a specimen. Following the initialstep 1302 in which the completion of the staining and other phases(e.g., one or more rinsing phases) is verified, in step 1304 the dryer 4directs a flow of air across the specimen. The drying process continuesin step 1306, until a signal is received from the control unit to stopthe drying. When the signal is received, the dryer stops the flow of airacross the specimen and the drying phase terminates at step 1308.

In general, machine 1 can be controlled to vary the temperature of theair, the flow rate, the duration of the applied air flow, and thephase(s) during specimen processing for drying the specimen 3. Forexample, after completing a staining phase, dryer 4 can direct a flow ofair at approximately 120° F. at a rate of 10 liters per minute for aperiod of 7 seconds across the specimen. Other air temperatures (e.g.,from ambient temperature up to 300° F.), air flow rates (e.g., one literper minute to 100 liters per minute), and air flow periods (e.g., from afew seconds to several minutes) can also be used.

Specimen Examination Systems

The automated specimen preparation machines and apparatus disclosedherein, including machine 1, can generally be used with, and/orincorporated into, larger specimen examination systems, such as thosedescribed in U.S. application Ser. Nos. 12/430,885 and 13/293,050, theentire contents of which are incorporated herein by reference. Forexample, FIG. 21 shows a schematic diagram that illustrates one possibleembodiment of a specimen examination system 2000. System 2000 includes aplatform 2100, a light receiving device 2200, a computer 2300, anapplicator 2400, a gas circulation device 2500, a light source 2600, adispenser 2800, a discharge device 2900, a slide labeler 3000, and slidelabel reader 3100. An advancer 2110 may be configured to receive one ormore slides or other substrates 2700. The advancer 2110 may be attachedto a surface, such as the top surface 2101, of the platform. Theadvancer 2110 may take the form of a belt, and the system may use amechanical arm, gravity, magnetism, hydraulics, gears, or otherlocomotion techniques to move substrate-mounted specimens along thesurface 2101 of the platform.

The platform 2100 may also include a feeder 2102 and a collector 2106for respectively feeding and collecting substrates 2700 (e.g., slides)from or to a stack or rack. Feeder 2102 may be equipped with a feederpropulsion mechanism 2103 (such as rubberized wheels) for pushing thespecimens onto advancer 2110. Alternatively, a mechanical arm could beused to grab substrates 2700 and place the substrates on the advancerdirectly. Alternate mechanisms to propel the substrates out of feeder2102 may be used such as magnets or hydraulics. The feeder may include asensor for determining how many slides are present. The sensor couldmeasure the weight of substrates 2700 for example to determine how manysubstrates are present. Collector 2106 can also include a sensor fordetermining how many substrates are present. The sensor can beconfigured to inform the computer 2300 when a preset number of specimenshave been analyzed, and/or can inform the computer of the receipt of aspecimen mounted on a substrate on an ongoing basis.

Light receiving device 2200 can be a microscope (such as brightfieldmicroscope), a video camera, a still camera, or other optical devicethat receives light. Embodiments that include a standard brightfieldmicroscope can also include an automated stage (e.g., a substrate mover2201) and an automated focus. In some embodiments, a microscope can beattached to a motorized stage and a focus motor attachment. Themicroscope can have a motorized nosepiece for allowing differentmagnification lenses to be selected under the control of computer 2300.A filter wheel can be used to enable the computer 2300 to automaticallyselect narrow band color filters in the light path. LED illumination canbe substituted for the filters, and the use of LEDs can reduce the imageacquisition time as compared to the time required for filter wheelrotation. For example, a 1600×1200 pixel FireWire® (IEEE1394 HighPerformance Serial Bus) camera can be used to acquire the narrow bandimages.

In some embodiments, light receiving device 2200 receives lightreflected from substrate 2700 and stores one or more images formed bythe reflected light. Alternatively, or in addition, in some embodiments,fluorescent emission from the specimen on the substrate can be detectedby light receiving device 2200.

In certain embodiments, light receiving device 2200 is configured toobtain transmission images of specimens on substrates. For example,light emission source 2600 can be positioned below the platform and maydirect light so that it passes through platform 2100 and substrate 2700into light receiving device 2200.

Light receiving device 2200 and any of the other components shown inFIG. 21 can be interfaced with the computer 2300 through links(2011-2014), which can provide energy to the component, provideinstructions from computer 2300 to the component, and/or allow thecomponent to send information to computer 2300. Links 2011-2014 can bewired links or wireless links.

Light receiving device 2200 may be capable of X, Y, and Z axial movement(in other embodiments, a motorized stage or substrate mover 2201 mayprovide X, Y, and Z movement). Light receiving device 2200 can includepan, tilt, and/or locomotive actuators to enable computer 2300 toposition light receiving device 2200 in an appropriate position. Lightreceiving device 2200 can include a lens 2210 that focuses incominglight.

Light receiving device 2200 can be selected to capture black and whiteand/or color images. In some embodiments, two or more light receivingdevices can be used to divide the processing time associated withcapturing the images. For example, a low magnification imaging stationcan be followed by a high magnification imaging station. Similarly, insome embodiments, system 2000, platform 2100, computer 2300, and/orlight receiving device 2200 can direct substrate mover 2201 to movesubstrate 2700 to ensure the capture and storage of one or more imagesof all, or most, of the cells on the substrate or on a specific portionof the substrate.

Computer 2300 can be a laptop, a server, a workstation, or any othertype of computing device. The computer can include a processor, adisplay 2320, an interface 2310, and internal memory and/or a diskdrive. Computer 2300 can also include software stored in the memory oron computer readable, tangible media such as an optical drive. Thesoftware may include instructions for causing the computer to operatelight receiving device 2200, applicator 2400, gas circulation device2500, platform 2100, advancer 2110, light source 2600, dispensers 2450and/or 2800, specimen preparation machine 1, or any component within orconnected to one of these components. Similarly, the computer isarranged to receive information from any of these components.

For example, the software may control the rate of dispersal ofsubstrates from the feeder 2102, and feeder 2102 may inform the computerabout the number of substrates present. In addition, computer 2300 canalso be responsible for performing the analysis of the images capturedby light receiving device 2200. Through the analysis process, thecomputer can be arranged and controlled to calculate the number of aspecific type of cell in a particular volume of blood, for example forblood, red cell, white cell, and platelet counts and other measured andderived components of the complete blood count such as: hemoglobincontent, red blood cell morphology, or white blood cell countdifferential could be calculated. The image analysis software cananalyze each individual field and sum the total red and white cellcounts. To calculate the total counts per microliter in a patient bloodsample, the number counted on the slide can be multiplied by thedilution ratio and volume of the sub-sample. Results of the counts,morphologic measurements, and images of red blood cells and white bloodcells from the slide may be shown on the display 2320.

In some embodiments, computer 2300 is configured to display numericaldata, cell population histograms, scatter plots, and direct assessmentsof cellular morphology using images of blood cells displayed on themonitor. The ability to display cellular morphology provides users ofsystem 2000 the ability to quickly establish the presence or absence ofabnormalities in cell morphology that may warrant preparing anadditional slide for manual review by an experienced technician or otherprofessional. The software can also provide the computer withinstructions to display images 2331 received from the light receivingdevice or may cause display 2330 to show the results 2332 (in perhaps achart or graph, for example) of an analysis of the images. Similarly,computer 2300 can be controlled to enumerate the number of cells of aspecific type in a particular blood volume or enumerate the number ofdamaged cells, cancerous cells, or lysed cells in a particular volume ofblood. The software enables the computer to perform the analysisprocess. The computer can use one or more magnifications during theanalysis.

Although shown as one component, computer 2300 can include multiplecomputers; a first computer can be used for controlling the componentsof system 2000, and a second computer can be used for processing theimages from light receiving device 2200. The various computers can belinked together to allow the computers to share information. Computer2300 can also be connected to a network or laboratory information systemto allow the computer to send and receive information to othercomputers.

In certain embodiments, applicator 2400 can include a syringe, a manualor motor driven pipettor, or a motor-controlled pump attached through atube to a pipette tip. Applicator 2400 applies a specimen to substrate2700 in controlled fashion. Exemplary features, attributes, and methodsof using applicator 2400 are disclosed, for example, in U.S. PatentApplication Publication No. US 2009/0269799. The specimen can includeone or more blood components, cells, tissue, or other biologicalcomponents.

Once the specimen has been applied to substrate 2700, the appliedspecimen is processed using machine 1. Machine 1 functions as describedherein to apply one or more stain solutions, fixative solutions, and/orother solutions to the specimen on the substrate.

In some embodiments, system 2000 can be configured to achieve minimaloverlapping between cells deposited on substrate 2700 by laying downnon-touching rows of cells from the tip of applicator 2400. Increasingviscosity of the diluted fluid or the type or amount of diluent mayaffect the width of the final settlement positions of specimen flowsfrom the applicator. By selecting a distance between rows to allow forthe typical variation in blood samples, all cells can be counted in allsamples.

Gas movement device 2500, which can be a separate device as shown inFIG. 21, or can be incorporated into machine 1 as discussed previously,can include a fan and/or may include other gas movement devices such asa compressor or a bellows for example. Gas movement device 2500 may beconnected directly to the computer 2300 or may be connected throughanother component such as platform 2100 or applicator 2400. The gasmovement device pushes gas (in some cases atmospheric air) across thesubstrate to control the rate at which substances on the substrate dry.Moving too much air too quickly (i.e., too high of a fan speed) acrossthe substrate can cause cells in the specimen to burst due to rapiddrying, and too little air too slowly (i.e., too low of a fan speed)across the substrate can cause the cells to dry too slowly and appear toshrink.

Computer 2300 can select and control the amount of air that moves acrossthe substrate in a period of time (i.e., the cubic feet or cubiccentimeters of air per second) based upon the distance the gas movementdevice is from the substrate, the type of fluid being analyzed, thewidth of the flows, the temperature of the gas (e.g., air), and theaverage thickness of the flows. Gas movement device 2500 can bepositioned so that the device directs gas so that the gas strikes thesubstrate at an angle of 30°-60° (e.g., 45°) for a period of about 15 to20 seconds. In some embodiments, computer 2300 can control humidity andtemperature settings in the vicinity of the system to allow the dryingprocess to occur without the use of a gas movement device 2500.

Light emission device 2600, and the various components thereof, aredescribed by way of example in U.S. Patent Application Publication No.US 2009/0269799. Various wavelengths of light can be generated by lightemission device 2600 and detected by light receiving device 2200. Forexample, wavelengths such as 415 nm are useful for obtaining ahemoglobin-only image for assessing RBC morphology and hemoglobincontent. Light emitted at 600 nm may be useful to provide high contrastimages for platelets and nuclei. Other wavelengths may be chosen inorder to best discriminate the colors of basophils, monocytes,lymphocytes (all shades of blue), eosinophils (red), and neutrophils(neutral color).

EXAMPLES

The disclosure is further described by the following examples, which arenot intended to limit the scope of the invention recited in the claims.

Example 1

FIG. 22 is a flow chart 1400 showing a series of exemplary steps forprocessing a specimen mounted on a substrate. The steps in flow chart1400 can be used to prepare a specimen for examination. Although thedescription of this process at times refers to specific steps havingspecific ranges or discloses steps occurring in a specific sequence,this description is intended to illustrate only one example process.With reference to FIG. 22, machine 1 is connected to a control system 5for commanding the operation of various machine components during theprocessing steps. In a specimen initiation step, a specimen 3 thatincludes red blood cells, white blood cells, and platelets from analiquot of blood is applied to a substrate 2 consisting of a glassmicroscope slide. This can be performed using a different station suchas one or more of the stations described in co-pending U.S. PatentApplication Publication No. 2008/0102006. In a positioning step 1402,substrate 2 containing specimen 3 is loaded onto substrate gripper 20Aof substrate arm 10A as shown in FIG. 7. Control system 5 instructssuction source 222 (step 1404) to evacuate air from the substrategripper 20A. Suction applied through suction ports 21 and 22 (step 1406)adheres the substrate 2 to the substrate gripper 20A during specimenprocessing. Control system 5 instructs (step 1408) the actuator 30A torotate the substrate 3 from an open position shown in FIG. 7 to aspecimen processing position shown in FIG. 9A. In the specimenprocessing position, specimen 3 faces the surface of platform 60A whilesubstrate 2 rests against offsets 70A-D shown in FIG. 8. The offsetsprevent the substrate 2 from making contact with the surface of platform60A. In this exemplary process, the separation 92 between thespecimen-containing surface of substrate 2 and the surface of platform60A is approximately 100 microns or 200 microns (e.g., 200 microns).

During a first fixation phase (step 1412, see also FIG. 15), a pumpapplies a fixative solution to the specimen 3 in step 1414. Pump 200Aconnected to fluid tube 54A shown in FIG. 8 propels a fixative solutioncomprising methanol from a fixative reservoir 210 through tube 54A, outport 44A, onto platform 60A, onto substrate 2, and into the separation92 between platform 60A and substrate 2. Pump 200A propels the fixativesolution from port 44A at a flow rate of 115 microliters per second fora two second period T1, thereby directing a total of 230 microliters ofthe fixative solution, V1, onto substrate 2.

Next, in a first agitation step 1416, control system 5 agitates thesubstrate by directing actuator 30A (step 1418) to raise the proximateedge of substrate 2 vertically a distance of 35 microns from thespecimen processing position and returning the specimen to its specimenprocessing position. Machine 1 repeats this agitation step four moretimes. The machine 1 completes the five agitation movements inapproximately ten seconds, T2, as shown in FIG. 23. After agitation, thecontrol system initiates a vacuum step 1420. A vacuum force of −0.10 psiis applied for one and a half seconds, T3, evacuating any residualfixative solution (step 1422) present in the separation, on theplatform, or on the substrate via ports 40A and 41A, and waste tubes 50Aand 51A. The evacuated fixative solution is collected in a wastecontainer 230 and/or 231.

Thereafter, in a second fixation phase including a second agitationstep, the foregoing steps of the first fixation phase and firstagitation step are repeated.

Following the fixing phase, control system 5 initiates (step 1424) afirst staining phase. In doing so, control system 5 directs the machine1 to stain the specimen (step 1426). Referring to FIG. 8 and theflowchart of FIG. 16, pump 201 connected to fluid tube 52A propels afirst stain solution comprising eosin Y from a stain reservoir 211A outport 42A, onto platform 60A, onto substrate 2, and into the separation92 between the platform 60A and substrate 2. Pump 201 dispenses thefirst stain solution through port 42A at a flow rate of 115 microlitersper second for a two second period, T4, thereby directing 230microliters of the first stain solution, V2, onto the substrate.

After applying a first stain solution to specimen 3, machine 1 performsa second agitation step 1428 by directing actuator 30A to raise, in step1430, the proximate edge of substrate 2 vertically a distance of 35microns from the specimen processing position and then return thespecimen to its specimen processing position. Control system 5 causesthe machine 1 to repeat this agitation step two more times and completethe three agitations over a period of approximately six seconds, T5, asshown in FIG. 23.

Next a second vacuum phase is initiated in step 1432. A vacuum of fivepsi applied for three seconds, T6, in step 1434 to evacuate any residualfirst stain solution present in the separation 92 or on the platform andsubstrate via ports 40A and/or 41A, and waste tubes 50A and 51A. Theevacuated first stain solution is collected in a waste container 230Aand/or 231 A.

After staining the specimen with the first stain solution includingeosin Y, machine 1 initiates a second staining phase in step 1436 usinga second stain solution including azure B and methylene blue. Pump 202connected to fluid tube 53A propels the second stain solution from astain reservoir through port 43A, onto platform 60A, onto substrate 2,and into the separation 92 between platform 60A and substrate 2 (step1438). Machine 1 dispenses the second stain solution through port 43A ata flow rate of 115 microliters per second for a two second period, T7,thereby directing a total of 230 microliters of the second stainsolution, V3, onto the substrate.

After applying stain to specimen 3, machine 1 initiates a thirdagitation phase in step 1440 by directing actuator 30A to raise theproximate edge of substrate 2 (step 1442) a distance of 35 microns fromthe specimen processing position and then return the specimen 3 to itsspecimen processing position. Machine 1 repeats this agitation stepthree more times. The machine completes the four agitation movementsover a period of approximately eight seconds, T8.

A third vacuum step 1444 is then initiated. A vacuum of five psi isapplied for two seconds, T9, to evacuate residual second stain solutionin step 1446 present in the separation or on the platform 60A andsubstrate 2 via ports 40A and/or 41A, and waste tubes 50A and/or 51A,after agitation. The evacuated second stain solution is collected in awaste container 230A and/or 231A.

Machine 1 then performs two rinse-agitation-vacuum phase sequences. Thefirst sequence of phases is initiated at step 1448 when control system 5instructs machine 1 to initiate a first rinse phase. A reservoir 213Acontaining a rinse solution is connected to a pump 203 and fluid tube55A. Pump 203 directs the rinse solution through wash tube 55A thatfeeds into port 45A, into the separation 92, and onto platform 60A andsubstrate 2 to rinse specimen 3 in step 1450. Alternatively, in someembodiments, rinse solution is directed through two or more of fluidports 42A to 45A. Pump 203 directs the rinse solution out of ports 45Aat a flow rate of 115 microliters per second for two seconds, T10,thereby directing a total of 230 microliters, V4, of water onto thesubstrate. Next, control system 5 initiates a fourth agitation phase instep 1452, directing actuator 30A (step 1454) to raise the proximateedge of substrate 2 vertically a distance of five microns from thespecimen processing position and returning the specimen to its specimenprocessing position. Control system 5 may direct the machine 1 to repeatthis agitation phase, and complete the two agitations in approximatelyfour seconds, T11.

Then, a vacuum phase is initiated in step 1456. A vacuum of five psiapplied for five and a half seconds, T12, in step 1458, evacuatesresidual rinse solution present in the separation 92 or on the platform60A and substrate 2 via ports 40A and/or 41A, and waste tubes 50A and/or51A after agitation.

Thereafter, in step 1460, control system 5 directs machine 1 to beginthe second rinse-agitation-vacuum phase sequence by initiating a secondrinse phase. A second rinse phase (steps 1460, 1462), a fifth agitationphase (steps 1464, 1466), and a fifth vacuum phase (steps 1468, 1470)are performed in the same manner as disclosed above for the firstrinse-agitation-vacuum phase. During the second rinse-agitation-vacuumphase, the amount of rinse solution, V5, and the processing times T13,T14, and T15 are generally the same as in the firstrinse-agitation-vacuum phase sequence.

After the specimen has been fixed, stained with a first stain solutionincluding eosin Y and a second staining solution including azure B andmethylene blue, and rinsed, machine 1 initiates a drying phase in step1472. Dryer 4 directs an air flow of approximately 120° at a 10liter-per-minute flow rate (step 1474) for an eight second period, T16,across the specimen.

Following completion of these steps, substrate 2 is returned to itsoriginal position in step 1476. In this step, actuator 30A rotatessubstrate 2 from the specimen processing position to the open positionas depicted in FIG. 7. Substrate 2 may then be removed by a substratemover, and a new substrate may be loaded for processing a new specimen.

As illustrated in the exemplary specimen processing steps describedabove and shown in FIG. 23, the systems and methods disclosed hereinprovide for more efficient specimen processing by consuming fewerreagents as compared to conventional specimen processing methodsincluding automated and manual specimen preparation techniques.Referring to FIG. 23, for example, machine 1 consumed less than one anda half milliliters of reagents for fixing, staining, and rinsing thespecimen during the exemplary processing steps (e.g., 460 microliters offixative solution+230 microliters of first stain solution+230microliters of second stain solution+460 microliters of rinsesolution=1380 microliters of reagents). In some embodiments, more orless than 1380 microliters of fluids can be used during specimenprocessing. For example, the amount of fluid used in processing aspecimen can be approximately 1150 microliters (e.g., by eliminating oneof the rinse phases).

The above-described fluid volumes relate generally to a spacing ofapproximately 100 microns between the substrate and platform. When thespacing between the substrate and platform is larger, greater volumes offluid are generally consumed during specimen processing. For example,when the spacing is approximately 200 microns, the total volume offluids consumed would have to be more than 1380 microliters.

More generally, the total volume of fluids consumed can be 500microliters or more (e.g., 520 microliters or more, 540 microliters ormore, 560 microliters or more, 580 microliters or more, 600 microlitersor more, 650 microliters or more, 700 microliters or more, 750microliters or more) and/or 2 mL or less (e.g., 1.5 mL or less, 1.4 mLor less, 1.3 mL or less, 1.2 mL or less, 1.1 mL or less, 1.0 mL or less,900 microliters or less).

Referring again to FIG. 23, the specimen preparation process iscompleted in slightly more than one minute (e.g., 13.5 seconds elapsedduring each of the fixing phases for a total of 27 seconds for fixing+11seconds elapsed during the first staining phase+12 seconds elapsedduring the second staining phase+23 seconds elapsed during the rinsephases+8 seconds elapsed during the drying phase=81 seconds totalelapsed time). In certain embodiments, specimen preparation can becompleted in more or less than 81 seconds. For example, specimenprocessing can be completed in 180 seconds or less (e.g., 150 seconds orless, 120 seconds or less, 90 seconds or less, 80 seconds or less, 70seconds or less, 60 seconds or less, 50 seconds or less, or 40 secondsor less).

Further, while the foregoing exemplary process describes processing timefor a single specimen, systems and methods for processing multiplesubstrates (e.g., machine 1 in FIG. 7, configured to process twosubstrates, and/or systems configured to process three or moresubstrates) are capable of processing more than 100 specimens per hour(e.g., between 60 specimens and 120 specimens per hour). Use of thesystems and methods disclosed herein in laboratory settings can resultin faster throughput on a per specimen basis, while consumption offluids (e.g., fixative, stain, and rinse fluids) is reduced compared toconventional automated systems and manual specimen preparationtechniques.

Example 2

The processing steps described above for Example 1 may be adjusted inother embodiments of the invention as follows. In addition, fixative,stains, and rinse solution formulations described herein can be used inthe following example processing steps.

During a first fixation phase (step 1412, see also FIG. 22), a pumpapplies a fixative solution to the specimen 3 in step 1414. Pump 200Aconnected to fluid tube 54A shown in FIG. 8 propels a fixative solutioncomprising methanol from a fixative reservoir 210 through tube 54A, outport 44A, onto platform 60A, onto substrate 2, and into the separation92 between platform 60A and substrate 2. Pump 200A propels the fixativesolution from port 44A at a flow rate of 115 microliters per second fora two second period T1, thereby directing a total of 230 microliters ofthe fixative solution, V1, onto substrate 2.

Next, in a first agitation step 1416, control system 5 agitates thesubstrate by directing actuator 30A (step 1418) to raise the proximateedge of substrate 2 vertically a distance of 35 microns from thespecimen processing position and returning the specimen to its specimenprocessing position. Machine 1 repeats this agitation step five moretimes. The machine 1 completes the six agitation movements inapproximately 12 seconds. After agitation, the control system initiatesa vacuum step 1420. A vacuum force of negative six psi is applied forone and a half seconds, T3, evacuating any residual fixative solution(step 1422) present in the separation, on the platform, or on thesubstrate via ports 40A and 41A, and waste tubes 50A and 51A. Theevacuated fixative solution is collected in a waste container 230 and/or231.

Thereafter, in a second fixation phase including a second agitationstep, the foregoing steps of the first fixation phase and firstagitation step are repeated.

Following the fixing phases, control system 5 initiates (step 1424) afirst staining phase. In doing so, control system 5 directs the machine1 to stain the specimen (step 1426). Referring to FIG. 8 and theflowchart of FIG. 16, pump 201 connected to fluid tube 52A propels afirst stain solution comprising eosin Y from a stain reservoir 211A outport 42A, onto platform 60A, onto substrate 2 including specimen 3, andinto the separation 92 between the platform 60A and substrate 2. Pump201 dispenses the first stain solution through port 42A at a flow rateof 115 microliters per second for a two second period, T4, therebydirecting 230 microliters of the first stain solution, V2, onto thesubstrate.

After applying a first stain solution to specimen 3, machine 1 performsa second agitation step 1428 by directing actuator 30A to raise, in step1430, the proximate edge of substrate 2 vertically a distance of 35microns from the specimen processing position and then return thespecimen to its specimen processing position. Control system 5 causesthe machine 1 to repeat this agitation step two more times and completethe three agitations over a period of approximately six seconds, T5, asshown in FIG. 23.

Next a second vacuum phase is initiated in step 1432. A vacuum ofnegative five psi applied for three seconds, T6, in step 1434 toevacuate any residual first stain solution present in the separation 92or on the platform and substrate via ports 40A and/or 41A, and wastetubes 50A and 51A. The evacuated first stain solution is collected in awaste container 230A and/or 231 A.

After staining the specimen with the first stain solution includingeosin Y, machine 1 initiates a second staining phase in step 1436 usinga second stain solution including azure B and methylene blue. Pump 202connected to fluid tube 53A propels the second stain solution from astain reservoir through port 43A, onto platform 60A, onto substrate 2,and into the separation 92 between platform 60A and substrate 2 (step1438). Machine 1 dispenses the second stain solution through port 43A ata flow rate of 115 microliters per second for a two second period, T7,thereby directing a total of 230 microliters of the second stainsolution, V3, onto the substrate.

After applying stain to specimen 3, machine 1 initiates a thirdagitation phase in step 1440 by directing actuator 30A to raise theproximate edge of substrate 2 (step 1442) a distance of 35 microns fromthe specimen processing position and then return the specimen 3 to itsspecimen processing position. Machine 1 repeats this agitation step twomore times. The machine completes the three agitation movements over aperiod of approximately six seconds, T8.

A third vacuum step 1444 is then initiated. A vacuum of negative six psiis applied for two seconds, T9, to evacuate residual second stainsolution in step 1446 present in the separation or on the platform 60Aand substrate 2 via ports 40A and/or 41A, and waste tubes 50A and/or51A, after agitation. The evacuated second stain solution is collectedin a waste container 230A and/or 231A.

Machine 1 then performs two rinse-agitation-vacuum phase sequences. Thefirst sequence of phases is initiated at step 1448 when control system 5instructs machine 1 to initiate a first rinse phase. A reservoir 213Acontaining a rinse solution is connected to a pump 203 and fluid tube55A. Pump 203 directs the rinse solution through wash tube 55A thatfeeds into port 45A, into the separation 92, and onto platform 60A andsubstrate 2 to rinse specimen 3 in step 1450. Alternatively, in someembodiments, rinse solution is directed through two or more of fluidports 42A to 45A. Pump 203 directs the rinse solution out of ports 45Aat a flow rate of 115 microliters per second for two seconds, T10,thereby directing a total of 230 microliters, V4, of water onto thesubstrate.

Next, control system 5 initiates a fourth agitation phase in step 1452,directing actuator 30A (step 1454) to raise the proximate edge ofsubstrate 2 vertically a distance of 35 microns from the specimenprocessing position and returning the specimen to its specimenprocessing position. Control system 5 then directs the machine 1 torepeat this agitation phase three more times, and complete the fouragitations in approximately eight seconds, T11.

Then, a vacuum phase is initiated in step 1456. A vacuum of five psiapplied for five and a half seconds, T12, in step 1458, evacuatesresidual rinse solution present in the separation 92 or on the platform60A and substrate 2 via ports 40A and/or 41A, and waste tubes 50A and/or51A after agitation.

Thereafter, in step 1460, control system 5 directs machine 1 to beginthe second rinse-agitation-vacuum phase sequence by initiating a secondrinse phase. A second rinse phase (steps 1460, 1462), a fifth agitationphase comprising six agitations completed in approximately 12 seconds,and a fifth vacuum phase (steps 1468, 1470) are performed in the samemanner as disclosed above for the first rinse-agitation-vacuum phase.During the second rinse-agitation-vacuum phase, the amount of rinsesolution, V5, and the processing times T13, T14, and T15 are generallythe same as in the first rinse-agitation-vacuum phase sequence. Inaddition, immediately prior to the vacuum phase, actuator 30A raises theproximate edge of substrate 2 a distance of 15-35 microns from thespecimen processing position. This increased separation betweensubstrate 2 and platform 60 improves evacuation of any residual fluidsin separation 92 during the final vacuum phase.

After the specimen has been fixed, stained with a first stain solutioncontaining eosin Y and a second staining solution containing azure B andmethylene blue, and rinsed, machine 1 initiates a drying phase in step1472. Dryer 4 directs an air flow of approximately 120° at a 10liter-per-minute flow rate (step 1474) for an eight second period, T16,across the specimen.

Following completion of these steps, substrate 2 is returned to itsoriginal position in step 1476. In this step, actuator 30A rotatessubstrate 2 from the specimen processing position to the open positionas depicted in FIG. 7. Substrate 2 may then be removed by a substratemover, and a new substrate may be loaded for processing a new specimen.

As illustrated in the example specimen processing steps described above,the systems and methods disclosed herein provide for more efficientspecimen processing by consuming fewer reagents as compared toconventional specimen processing methods including automated and manualspecimen preparation techniques. Referring to Example 2, machine 1consumed less than one and a half milliliters of reagents for fixing,staining, and rinsing the specimen during the exemplary processing steps(e.g., 460 microliters of fixative solution+230 microliters of firststain solution+230 microliters of second stain solution+460 microlitersof rinse solution=1380 microliters of reagents). In some embodiments,more or less than 1380 microliters of fluids can be used during specimenprocessing. For example, the amount of fluid used in processing aspecimen can be approximately 1150 microliters (e.g., by eliminating oneof the rinse phases) or less than 1,000 microliters (e.g., by furthereliminating one of the fixative phases).

With respect to FIG. 23, for Example 1, machine 1 consumed less than onemilliliter of reagents for fixing, staining, and rinsing the specimenduring the exemplary processing steps (e.g., 140 microliters of methanolfixative+140 microliters of fluorescein dye+140 microliters of thiazindye+280 microliters of rinse solution=700 microliters of reagents). Insome embodiments, more or less than 700 microliters of fluids can beused during specimen processing. For example, the amount of fluid usedin processing a specimen can be approximately 560 microliters (e.g., byeliminating one of the rinse phases).

In general, the total volume of fluids consumed can be 500 microlitersor more (e.g., 520 microliters or more, 540 microliters or more, 560microliters or more, 580 microliters or more, 600 microliters or more,650 microliters or more, 700 microliters or more, 750 microliters ormore) and/or 2 mL or less (e.g., 1.5 mL or less, 1.4 mL or less, 1.3 mLor less, 1.2 mL or less, 1.1 mL or less, 1.0 mL or less, 900 microlitersor less).

Referring to FIG. 23 and Example 1, the specimen preparation process iscompleted in slightly more than one minute (e.g., 13.5 seconds elapsedduring the fixing phase+11 seconds elapsed during the fluorescein dyephase+12 seconds elapsed during the thiazin dye phase+23 seconds elapsedduring the rinse phases+8 seconds elapsed during the drying phase=67.5seconds total elapsed time). In certain embodiments, specimenpreparation can be completed in more, as in Example 2, or less than 67.5seconds. For example, specimen processing can be completed in 180seconds or less (e.g., 150 seconds or less, 120 seconds or less, 90seconds or less, 80 seconds or less, 70 seconds or less, 60 seconds orless, 50 seconds or less, or 40 seconds or less).

Further, while the foregoing exemplary process describes processing timefor a single specimen, systems and methods for processing multiplesubstrates (e.g., machine 1 in FIG. 7, configured to process twosubstrates, and/or systems configured to process three or moresubstrates) are capable of processing more than 100 specimens per hour(e.g., between 60 specimens and 120 specimens per hour). Use of thesystems and methods disclosed herein in laboratory settings can resultin faster throughput on a per specimen basis, while consumption offluids (e.g., fixative, stain, and rinse fluids) is reduced compared toconventional automated systems and manual specimen preparationtechniques.

Example 3

For each of the series of experiments in Table 1, blood samples wereprepared using the sample preparation technique as described, forexample, in U.S. Publication No. US20090269799. The samples were thenprocessed by fixing, staining, and rinsing generally according toExample 1, and the flow rate was 115 μL/s for two seconds for each ofthe fixative solution, stain solutions, and rinse solutions. For eachset of experiments listed below, substrates (e.g., microscope slides)were prepared from at least five blood samples. Next, the processedsamples were manually evaluated and the quality of the sample stainingand preparation (e.g., overall uniformity of staining, color,differentiation of cellular features, presence/absence of debris inbackground, etc.) was assessed under a microscope, using at least 10×magnification. Manual evaluation was performed to compare the stain andsample preparation quality of the sample specimen versus the stain andsample prep quality of a control specimen. A “rolling control condition”was typically employed, where a previous formulation that providedoptimal staining and sample preparation was used as the control forcomparing new adjustments to the various formulations. Thus (unlessotherwise specified below), any given row in Table 1 typicallyrepresents the control condition for the series of experimentssummarized in the following row. For a limited number of samples andafter manual review, the samples were processed on an imager asdescribed in US20090269799 to test how, if at all, the formulationsimpacted the imager's ability to classify the five types of WBCs presentin the sample. These results are reported in Table 2.

TABLE 1 Formulation assessment experiments. Experiment, in Range/chronological Property parameter order Formulation Tested testedObservations/Results 2A Fixative Dye Azure B Prior to this series ofexperiments, less- addition than-optimal staining of basophils wasobserved (e.g., insufficient differentiation between nucleus andcytoplasmic granules). Adding dye to the fixative composition showedsignificant improvement in basophil staining (e.g., observedorange-reddish cytoplasmic granule staining). This fixative formulationremained constant until experiment 2L. 2B Rinse MeOH 0%, 5% The 5% MeOHin rinse solution showed Conc slightly improved evacuation of rinsesolution from the platen as compared to a rinse solution without MeOH.2-1 See Classification Results, Table 2. 2C Blue Stain pH 5.85-7.86Selected pH 6.5 for Blue Stain solution (containing Azure B, but notMethylene Blue) as a result of this series of experiments. Higher pHvalues provided less than optimal staining (e.g., cytoplasm stained toodark, neutrophils stained too blue). HEPES buffer was used to achievedesired pH. 2-2 See Classification Results, Table 2. 2D Blue Stain Tween20 0.01%-0.1%  0.1% Tween 20 was ultimately selected. Conc The blue andred stain formulations had an improved ability to uniformly stain thesample, as well as decreased risk of precipitation and non-specificbinding of stain. 2E Red Stain Tween 20 0.01%-0.5%  0.1% Tween 20 wasselected in the first Conc and second stain formulations. Theformulations had an improved ability to uniformly stain the sample, aswell as decreased risk of precipitation and non- specific binding ofstain. 2F 50/50 Blue Antimicrobial ProClin 300 ® 2G Red StainAntimicrobial ProClin 300 ® 2H Rinse Antimicrobial Benzalkonium ProClin300 ® was selected as the chloride, antimicrobial agent for the rinsesolution ProClin (as well as red and blue stains as noted in 300 ®previous two rows) over benzalkonium chloride due to benzalkoniumchloride's tendency to cause precipitation in the rinse solution. 2I RedStain Eosin 0.75 g/L-1.25 g/L Experiments were performed to evaluateConc staining WBC cytoplasms, particularly in immature granulocytes.Previous formulations, before an eosin concentration of 0.75 g/L wasselected, caused excessive redness in cytoplasms. 2J Blue StainMethylene  0%-70% Various relative proportions of Azure B blue Conc andMethylene Blue were tested in a blue staining solution. A 50/50formulation showed improved staining in lymphocyte and monocytecytoplasms, and was found to be a significant contributor to solving theexcessively red cytoplasm staining issue described in the preceding row.Azure B was also generating a red component to cytoplasmic staining,particularly lymphocytes and monocytes, which ultimately led toselection of 50% methylene blue. 2K 50/50 Blue pH 6.5-7.0 Using a 50/50azure B/methylene blue stain formulation as the control, the pH of theblue stain solution was varied. At pH 7.0 for the blue stain, slightimprovement (i.e., darker) on platelet staining was observed. The 50/50Blue stain formulation remained constant until experiment 2W. 2LFixative Tween 20   0%-0.5% 0.1% Tween 20 was used in fixative Concsolution. Using various points within the range, 0.1% Tween 20 showedbetter stain consistency throughout the sample deposited on the slide.Without the detergent, the stain would not flow evenly across stainingplaten, often leaving an unstained spot in the middle of the sample. 2MFixative pH 5.0-8.0 A series of experiments was conducted to verify thatpH 7 was optimal for the fixative. A pH of 6.5 or less showed pooroverall staining appearance. 2N Rinse pH 4.0-8.0 The pH of the rinse wasvaried to evaluate impact on staining quality and ultimately a pH of 6.8was selected. Higher pH caused bluer RBCs. Lower pH caused excessivelyred RBCs. 2-3 See Classification Results, Table 2. 2O Rinse Tween 200.01%-0.1%  0.1% Tween 20 concentration for the rinse Conc solution wasre-evaluated and re- confirmed. 2P Red Stain pH 5.7-7.7 The pH of thered stain was varied to determine an optimal pH for cytoplasmicstaining. pH 6 was selected. 2Q Red Stain MeOH 70% 70% MeOH in a redstain formulation at Conc pH 6 and pH 3.9 were evaluated to determinewhether the formulation could increase processing speed and improveoverall appearance and color of prepared samples. 70% MeOH at pH 6showed poor overall staining. 70% MeOH at pH 3.9 showed marginal overallstaining and saturated RBCs. 2R 50/50 Blue Dye Conc 0.5 g/L-1.0 g/LConcentrations of about 0.5 g/L for both Azure B and Methylene Bconcentrations in blue stain were reconfirmed. 2S Red Stain Bufferphosphate As a result of this series of experiments, buffers, BisTrisbuffer was selected over phosphate BisTris buffers for the red stainformulation. Without wishing to be bound by any theory, it is believedthat in some embodiments, the phosphate buffers can act as growth mediafor microorganisms. 2T Red Stain pH 5.5-6.0 A pH of 6.0 was reconfirmedfor the red w/BisTris stain formulation. 2U Red Stain pH 5.0-6.0 A pH of6.0 was reconfirmed for the red w/BisTris, stain formulation. NaCl 2VRed Stain NaCl   0%-0.6% Various concentrations of NaCl were testedw/BisTris Conc and 0.4% NaCl was selected for the red stain formulation.NaCl can decrease hemolysis and debris in a specimen's background. Inaddition, NaCl can minimize non-specific binding of stain. Withoutwishing to be bound by any theory, it is believed that a change from aphosphate to BisTris buffer may have contributed to debris present inthe specimen's background. 2W 50/50 Blue NaCl   0%-0.2% Variousconcentrations of NaCl were tested Conc and 0.2% NaCl was selected forthe blue stain formulation. Without wishing to be bound by any theory,it is believed that NaCl can decrease hemolysis and debris in aspecimen's background. In addition, NaCl can minimize non-specificbinding of stain. 2X 50/50 Blue Glycol 1% With respect to experimentsdescribed in propylene, this row and next two rows, 1.0% ethyleneethylene, glycol was used in fixative and red stain PE glycol solutions,as too little chromatin texture was observed in blast cells whenobserved through manual review. Ethylene glycol was not added to theblue stain solution because it produced no discernible effect on blastcells or otherwise. 2Y Fixative Glycol 1% propylene, ethylene, PE glycol2Z Red Stain Glycol 1% propylene, ethylene, PE glycol 2AA 50/50 BlueBuffer HEPES Different buffers were tested in a blue stain Sodiumformulation. BisTris was selected as a Salt, buffer, as it showedimproved compatibility BisTris (i.e., less reactions) with red stainsolution. 2BB Rinse Buffer MES, HEPES Sodium Salt/Acid, BisTris 2CCRinse PEG Conc 0%-1% 0.5% polyethylene glycol was selected for the rinsesolution, based on general overall appearance of sample and its abilityto fix prepared samples. 0.5% formulation reduced the amount of residueleft on the slide after evacuating the rinse solution as compared torinse solution containing 1% polyethylene glycol. 2DD Fixative Ethylene0%-5% 1% ethylene glycol in the fixative solution Glycol was confirmedby looking at samples Conc treated with 1, 2 and 5% ethylene glycolfixative solutions. Confirmed by manual review. 2EE Red Stain Ethylene0%-5% 1% ethylene glycol in the red stain solution Glycol was confirmedby looking at samples Conc treated with 1, 2 and 5% ethylene glycol redstain solutions. Confirmed by manual review. 2FF Rinse All 100%, 20% Evaluated overall sample appearance and components staining uniformitythrough a series of experiments by comparing samples prepared withprevious rinse formulation and samples prepared with a 20% rinseformulation (i.e., reducing all components to one-fifth of previousformulation). 20% rinse formulation showed significant improvement overprevious formulation by (i) reducing the extent of dried rinse film andreside left on sample, (ii) correcting tendency for sample to appearredder and diluting blue stain, (iii) diminishing stain spotting onsample and areas containing excessively red nuclei, and (iv) refiningoverall sample appearance and staining uniformity.

The WBC classifier results listed in Table 2 compare the automatedmachine WBC classification results as described in U.S. Publication No.US20090269799, herein incorporated in its entirety, against a manualclassification of WBCs in the sample. The percentages reflect anautomated machine classifier accuracy compared to manual review andclassification of WBCs in the sample. For each of the three experiments(2-1, 2-2, and 2-3), a minimum of ten samples prepared usingthen-current fixative and staining solutions were processed on thesystem and compared to a manual review of WBCs in each sample. Eachmanual review consisted of a WBC differential performed on at least 100WBCs in the sample.

TABLE 2 White blood cell (“WBC”) classification results. WBC Cell Type2-1 2-2 2-3 Lymph 60.3% 93.7% 97.2% Mono 83.3% 88.2% 94.3% Neut 89.2%97.6% 99.1% Baso NA NA 100.0% Eo 83.7% 94.9% 99.0%

Example 4

Dozens of commercially available hematology fixative and stainingproducts were tested in the systems described herein; such products weregenerally unsuitable for use with the cell identifying, counting, andclassification systems described herein. For example, the stainingdarkness achieved with these products was compared with that achievedwith the formulations described herein. In general, the products testeddid not produce the optimal sample preparation results (e.g., did notstain samples as darkly or as uniformly) as the formulations describedherein. The commercially available products, for example, producedlightly stained nuclei and cytoplasms when compared with theformulations described herein.

OTHER EMBODIMENTS

It is to be understood that while the invention has been described inconjunction with the detailed description, the foregoing description isintended to illustrate and not limit the scope of the disclosure, whichis defined by the scope of the appended claims. Other aspects,advantages, and modifications are within the scope of the followingclaims.

All references, such as patent applications, publications, and patents,referred to herein are incorporated by reference in their entirety.

What is claimed is:
 1. A cytological fixative solution consisting of asingle thiazine dye; a surfactant; a buffering agent; methanol; and anagent selected from the group consisting of ethylene glycol, propyleneglycol, polyethylene glycol, and any combination thereof.
 2. Thecytological fixative solution of claim 1, wherein the thiazine dye isAzure B.
 3. The cytological fixative solution of claim 1, wherein thethiazine dye is present in an amount of from about 0.5 to about fiveg/L.
 4. The cytological fixative solution of claim 1, wherein thesurfactant is present in an amount of from about 0.05% to about 0.5% byvolume.
 5. The cytological fixative solution of claim 1, wherein thesurfactant is present in an amount of from about 0.05% to about 0.3% byvolume.
 6. The cytological fixative solution of claim 1, wherein thesurfactant is selected from the group consisting of non-ionic, cationic,anionic, and zwitterionic surfactants.
 7. The cytological fixativesolution of claim 6, wherein the non-ionic surfactant is polysorbate 20.8. The cytological fixative solution of claim 7, wherein polysorbate 20is present in an amount of from about 0.5 mL/L to about 5.0 mL/L.
 9. Thecytological fixative solution of claim 1, wherein the buffering agent isselected from the group consisting of bis-tris, phosphate, HEPES, MES,Tris, and any combination thereof.
 10. The cytological fixative solutionof claim 9, wherein a 1:10 dilution of the solution in water has a pH offrom 6 to 8 and HEPES is present in an amount of from about 0.5 mM toabout 10 mM.
 11. The cytological fixative solution of claim 9, wherein a1:10 dilution of the solution in water has a pH of from 6.7 to 7.3 andHEPES is present in an amount of from about 0.5 mM to about 10 mM. 12.The cytological fixative solution of claim 1, wherein the agent isethylene glycol.
 13. The cytological fixative solution of claim 1,wherein the agent is present in an amount of from about 0.5 to about 5%by volume.
 14. The cytological fixative solution of claim 1, wherein a1:1000 dilution of the fixative solution in water has a UV absorbance offrom about 0.1 to about one at a peak wavelength of from about 640 toabout 650 nm.
 15. The cytological fixative solution of claim 1, whereinthe surfactant is non-ionic.
 16. A cytological fixative solutionconsisting of a single thiazine dye in an amount of from about 0.5 g/Lto about 5.0 g/L; about 0.5 mL/L to about 5.0 mL/L polysorbate 20; about5 mL/L to about 50 mL/L ethylene glycol; about 0.10 g/L to about 10 g/LHEPES sodium salt; and methanol.